Biomedical subjects

S O Andersen

Publications and source records attributed to S O Andersen.

At least 19 recordsLinked to original sources

PubMed2003

Characterization and cDNA cloning of three major proteins from pharate pupal cuticle of Manduca sexta.

Three proteins, MsCP20, MsCP27 and MsCP36, that are secreted in greatest quantity into the pharate pupal cuticle of Manduca sexta ( Hopkins et al., 2000) were purified and their amino acid sequences determined by mass spectrometry and Edman degradation. Although these proteins become sclerotized and insoluble in the pupal exoskeleton, their sequences contain features characteristic for proteins occurring in less sclerotized pliable cuticles, such as arthrodial membranes and soft larval cuticles. These proteins carry a secondary modification attached to a threonine residue, presumably an O-linked sugar moiety. cDNA clones of the genes for MsCP20, MsCP27 and MsCP36 were constructed from pharate pupal integument RNA. Close agreement was found between the amino acid sequences determined by Edman degradation and sequences deduced from the cDNA clones. The molecular masses determined by protein sequencing for MsCP20, MsCP27, and MsCP36 were 17713, 17448, and 29582 Da, respectively, in close agreement with the masses deduced from the corresponding cDNA clones (17711, 17410, and 29638 Da). Temporal expression analysis indicates that MsCP20 and MsCP36 transcripts are present at low levels early in the fifth larval stadium, followed by a large increase in abundance prior to pupal ecdysis. MsCP27 was not detected during development of the fifth larval instar, but its transcript, like those of MsCP20 and MsCP36, increased to a peak level just before pupal ecdysis. Only the MsCP36 transcript was detected in adults. These results support the hypothesis that these proteins are synthesized by the epidermis and are subsequently deposited into the cuticle during the larval-pupal transformation of M. sexta where they become sclerotized in the formation of pupal exocuticle.

Amino Acid Sequence↗

PubMed2002

Characteristic properties of proteins from pre-ecdysial cuticle of larvae and pupae of the mealworm Tenebrio molitor.

Proteins extracted from the cuticle of pharate larvae and pupae of the mealworm Tenebrio molitor are more soluble at low temperatures than at higher temperatures, a behaviour characteristic of hydrophobic proteins. When the temperature of an unfractionated cuticular extract is raised from 4 to 25 degrees C the solution becomes turbid, droplets of a heavy, protein-rich phase are formed, which gradually settles, leaving an upper protein-poor phase, indicating that the aggregation process is a coacervation. The aggregation of the dissolved cuticular proteins is influenced by changes in temperature, pH, and ionic strength. The process has been studied by measuring development of turbidity in unfractionated cuticular extracts and in solutions of three purified proteins from Tenebrio pharate larvae and pupae (TmLPCP-A1a, TmLPCP-E1a, and TmLPCP-G1a), while temperature, pH or ionic strength of the solutions were varied. Protein aggregation was also studied by determination of changes in fluorescence intensity, when the hydrophobicity probe, 8-anilinonaphthalenesulfonic acid (ANS) was added to solutions of the cuticular proteins. Only when the protein solutions had developed a measurable turbidity was an increase in ANS-fluorescence observed, indicating formation of tightly packed clusters of hydrophobic amino acid residues during aggregation. The temperature range for aggregation depends upon protein concentration: the higher the concentration the lower and more narrow is the temperature range within which aggregation occurs. The tendency for the individual cuticular proteins to aggregate is most pronounced near their isoelectric points, and most of the cuticular proteins have alkaline isoelectric points. The influence of salts on the tendency of the proteins to aggregate varies among the proteins and depends upon how close they are to their isoelectric point. A solution containing both protein TmLPCP-A1a and TmLPCP-E1a becomes more turbid and develops a more intense ANS-fluorescence when warmed from 10 to 30 degrees C than corresponding to the sum of measurements performed on separate solutions of the two proteins, indicating that the two proteins interact during aggregation. The Tenebrio larval/pupal cuticular proteins are characterized by an abundance of hydrophobic amino acid residues, and especially their contents of alanine and proline are high. The behaviour of the cuticular proteins in solution resembles that of another hydrophobic protein, tropoelastin, and it seems reasonable to suggest that similar interactions govern the folding and aggregation of the peptide chains in the two types of proteins. The proline and alanine rich chain segments in the pharate cuticular proteins are suggested to form a series of beta-turns and to fold into a relatively open structure at low temperatures, giving water access to the hydrophobic residues and making the proteins water soluble. At increased temperatures the structure of the ordered water layer surrounding the hydrophobic groups breaks down, and the peptide chains tend to collapse into a more closed structure and to interact more tightly with hydrophobic regions in neighbouring molecules. In dilute solutions in the test tube this results in aggregation and precipitation of the proteins; in intact, pharate cuticle at ambient temperatures the proteins will preferably be in an aggregated, easily dissociated state. Accordingly, small changes in intercuticular pH and ionic strength can produce pronounced changes in the mechanical properties of unsclerotized solid cuticle by interference with protein interactions, in agreement with reports that some cuticles undergo plasticization during and/or immediately after ecdysis.

Animals↗

PubMed2001

Purification and sequence determination of a yellow protein from sexually mature males of the desert locust, Schistocerca gregaria.

A yellow protein from abdominal cuticle of the desert locust, Schistocerca gregaria, has been purified and its amino acid sequence determined. The yellow color comes from bound carotene, the protein is only deposited in the epidermis and cuticle of male locusts during their sexual maturation, and the deposition is dependent upon a sufficiently high titer of juvenile hormone. The sequence of the protein is atypical for a cuticular protein, but it has some similarity to a putative juvenile hormone binding protein from Manduca sexta. It is suggested that the protein is involved in the transport of carotenes from internal tissues to epidermis and cuticle of the locust.

Amino Acid Sequence↗

PubMed2001

Matrix proteins from insect pliable cuticles: are they flexible and easily deformed?

Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk moth larvae, Hyalophora cecropia, were studied in solution by means of a fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in non-polar media than in polar media. An intense ANS-fluorescence was observed in the presence of the cuticular proteins at pH-values close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denaturants were added. Aggregation and eventual precipitation of both H. cecropia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A decreased ANS-fluorescence was observed at salt concentrations too low to cause visible aggregation of the H. cecropia proteins, probably due to weakened electrostatic interactions between chain segments, but such a decrease was not observed for the locust proteins. The changes in intensity of ANS-fluorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are exposed to the solvent, when either hydrophobic interactions or electrostatic attractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are available for protecting ANS-molecules from the quenching effects of water. The effects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as hen egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed hydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation corresponding to that of globular proteins. The behaviour of the cuticular proteins resembles that described for unordered, randomly coiled, thermally agitated polymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cuticular proteins are in an unordered, random structure both when the proteins are in solution and when present in the intact cuticle; probably only the chain regions involved in binding the proteins to chitin will have a well-defined spatial organisation.

Anilino Naphthalenesulfonates↗

PubMed2001

Tentative identification of a resilin gene in Drosophila melanogaster.

A search of the Drosophila genome for gene products with similarities to the amino acid sequences of three tryptic peptides from locust (Schistocerca gregaria) resilin gave two positive results: gene products CG15920 and CG9036. In both conceptual translation products a 62-residue region is present, which is identical to the resilin peptides in 29 positions. Gene product CG15920 has an amino acid composition closely resembling that of resilins from various insect species, and it has an N-terminal signal peptide sequence indicating that it is an extracellular protein. The 62-residue region shows similarity to the RR-2 sequence, which is common for a number of matrix proteins from insect solid cuticle. The N- and C-terminal regions flanking the 62-residue in CG15920 are dominated by 18 repeats of a 15-residue sequence and 11 repeats of a 13-residue sequence, respectively. The structures of the repeats predict that the peptide chain will fold in an irregular, extended beta-spiral, resembling the structures suggested for mammalian elastin and spider flagelliform silk, two materials which, like resilin, possess long-range elasticity. Accordingly, we suggest that gene product CG15920 is a Drosophila resilin precursor.

Amino Acid Sequence↗

PubMed2000

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer.

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.

Amino Acid Sequence↗

PubMed1999

Exoskeletal proteins from the crab, Cancer pagurus.

Twelve proteins from calcified regions and five from flexible regions (arthrodial membranes) of the exoskeleton of Cancer pagurus have been purified and sequenced. One of the proteins from calcified exoskeleton is identical to one of the arthrodial membrane proteins. Several of the proteins from the calcified regions resemble proteins from corresponding regions of the exoskeleton of the lobster, Homarus americanus, in containing either two or four copies of an 18-residue sequence motif, which so far has been found only in crustacean calcified exoskeletons. The proteins obtained from the flexible arthrodial membranes resemble the proteins from lobster arthrodial membranes, and the similarities are shared with a number of proteins from flexible cuticles in insects, indicating that the common features in these proteins may be important for the mechanical properties of the materials in which they occur.

Amino Acid Sequence↗

PubMed1998

Primary structure of two major cuticular proteins from the migratory locust, Locusta migratoria, and their identification in polyacrylamide gels by mass spectrometry.

The complete amino acid sequence has been determined for two proteins, LmACP21 and LmACP22, which are prominent components of adult pharate cuticle from the migratory locust, Locusta migratoria. The proteins have relative molecular masses (Mr) of 16853 and 16879, respectively. They were purified by standard chromatographic methods, and the primary structures were determined by combined use of mass spectrometry and automatic Edman degradation. The proteins are characterized by the presence of a conserved, hydrophilic central sequence with pronounced similarity to sequences reported for cuticular proteins from other insect species, while the N- and C-terminal regions are dominated by the amino acids alanine, valine and proline. The electrophoretic identity of the two proteins was confirmed by matrix assisted laser desorption ionization mass spectrometry (MALDIMS) of the electroeluted LmACP21/22 proteins from a two-dimensional electrophoresis gel. The mass spectrometric analysis established the presence of additional proteins in close proximity to the LmACP21/22 gel spot. One of these proteins, Mr 16134, was identified as LmACP18, and enzymatic digestion indicated that it is structurally closely related to LmACP21 and LmACP22.

Amino Acid Sequence↗

PubMed1998

Characterization of exoskeletal proteins from the American lobster, Homarus americanus.

Proteins from the calcified exoskeleton of the lobster, Homarus americanus, were extracted and separated by two-dimensional gel-electrophoresis. Electroblotting the proteins onto polyvinylidene difluoride (PVDF) membranes followed by sequence determination gave 16 N-terminal amino-acid sequences and revealed that further eight proteins were N-terminally blocked. The relative molecular mass, M(r), was obtained for most of the electrophoretically separated proteins by means of matrix-assisted laser desorption mass spectrometry (MALDIMS) after electroelution from Coomassie-stained two-dimensional polyacrylamide gels. Eleven proteins were purified from extracts of the exoskeleton by low pressure ion exchange chromatography and reversed-phase high performance chromatography, and their sequences were determined by combined use of Edman degradation and mass spectrometry. Good agreement was obtained between the M(r)-values measured by mass spectrometry and those calculated from the sequences. Five of the sequenced proteins contain two copies of a previously observed 18-residue sequence motif, while a couple of the remaining sequences show similarity to sequences of exoskeletal proteins from shrimps and spiders. Only limited similarity to insect cuticular proteins was observed.

Amino Acid Sequence↗

PubMed1998

Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus.

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses approximately 12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.

Acrylic Resins↗

PubMed1997

[Prescriptions, prescription-writing errors and patient safety].

The aim of the study is to contribute to knowledge about prescribing-writing errors on prescriptions from hospitals. One hundred and seventy prescriptions, prescribed from Copenhagen University Hospital, were evaluated in a project at Copenhagen University Hospital Pharmacy. Prescriptions were collected at two private pharmacies in Copenhagen during a two-week period in October 1995. The prescriptions contained an average of 1.5 errors and only 11% of the prescriptions were faultless. The three most frequent errors of omission were code of department, inadequate identification of the physician and indications of use. It is often difficult to reach the physician at the hospital, but the first step is to ease the identification of physician and ward. This project confirms that indication of use is a frequent error of omission.

Denmark↗

PubMed1997

Cuticular proteins from the lobster, Homarus americanus.

The urea-extractable proteins from calcified regions of intermoult cuticle of the lobster, Homarus americanus, have been separated by two-dimensional electrophoresis, showing that the extracts contain a large number of proteins. The major proteins have isoelectric points between 4 and 9, and their apparent molecular weights are between 5 and 30 kDa. Two of the proteins have been purified by a combination of ion-exchange chromatography, gel-filtration and RP-HPLC, and their complete amino acid sequences were determined by a combination of mass spectrometry and automated Edman degradation. Although they were purified from a single animal, both proteins were obtained as two isoforms. The isoforms of the smaller protein (HaCP4.6) differed only in a single position (phenylalanine/isoleucine), and the isoforms of the larger protein (HaCP11.6) differed in two positions (valine/isoleucine and glutamine/lysine). HaCP11.6 is N-terminally blocked by a pyroglutamate residue. Variants of an 18-residue motif are a characteristic feature of both sequences: it occurs twice in HaCP4.6 and four times in HaCP11.6. Comparison of the sequence to sequences published for cuticular proteins from other arthropods shows that the repeated motif is also present in proteins from the exoskeleton of the Bermuda land crab, Gecarcinus lateralis, but not in the single shrimp protein (Pandalus borealis) sequenced so far. The amino acid compositions of the lobster proteins are similar to that of flexible cuticles in locusts, but no convincing sequence similarities were found between the lobster proteins and cuticular proteins from locusts or other insects.

Amino Acid Sequence↗

PubMed1997

Cuticular proteins from the giant cockroach, Blaberus craniifer.

The extractable proteins from selected cuticular regions of nymphs and adults of the cockroach, Blaberus craniifer, have been compared by two-dimensional gel-electrophoresis. Only minor differences in protein patterns were observed when nymphal and adult pre-ecdysial cuticles (presumptive exocuticle) were compared, whereas the pattern obtained from nymphal mid-instar cuticle (mainly endocuticle) differed markedly from that obtained from mature adult cuticle. The pattern obtained from nymphal mid-instar cuticle depended upon the specific cuticular region analysed, but the differences within a stage were, to a large extent, quantitative and not qualitative. Seven nymphal endocuticular proteins have been purified to near homogeneity, and the complete amino acid sequence has been determined for three of them. One of the proteins, Bc-NCP1, contains a 16-residue motif repeated three times and containing a disulphide bridge. Protein Bc-NCP2 has a twice repeated motif in common with a pupal protein from Bombyx mori, and Bc-NCP4 contains a twice-repeated sequence of nine residues and is moreover characterized by an unusual high content of valine (22.0%). None of the protein sequences shows significant similarities to the sequences determined for locus endocuticular proteins, except that they all have pyroglutamate as the N-terminal residue.

Amino Acid Sequence↗

PubMed1997

Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor.

Complete amino acid sequences have been determined for six larval-pupal cuticular proteins from Tenebrio molitor. The sequenced proteins are major components in both larval and pupal cuticle, and both basic and slightly acidic proteins are represented. The proteins show pronounced similarities to some of the proteins sequenced from other insect cuticles. Three slightly acidic larval-pupal Tenebrio cuticular proteins contain a 66-residue central, hydrophilic region, resembling regions in cuticular proteins from insect species of four different orders (Coleoptera, Diptera, Lepidoptera and Orthoptera), and three basic proteins from larval-pupal Tenebrio cuticle have a 51-residue hydrophilic region in common with two proteins from cuticle of pharate adult locusts (Locusta migratoria). The Tenebrio larval-pupal cuticular proteins are also similar to locust adult cuticular proteins, by frequent occurrence of the short sequence motif Ala-Ala-Pro-Ala/Val. The pronounced sequence similarities between cuticular proteins from different insect orders indicate that the conserved regions are functionally important.

Amino Acid Sequence↗

PubMed1995

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.

Amino Acid Sequence↗

PubMed1995

Comparison of larval and pupal cuticular proteins in Tenebrio molitor.

Protein extracts from pupal and larval pharate cuticle from the meal worm, Tenebrio molitor, gave nearly identical patterns by two-dimensional electrophoresis and by ion-exchange chromatography. The main components in the cuticular extracts from the two metamorphic stages were also identical with respect to molecular mass according to electrospray ionization mass spectrometry. The complete amino acid sequence for one of the pupal cuticular proteins was determined; according to partial amino acid sequences and the mass spectrometric peptide map for the corresponding larval cuticular protein, it was concluded that the larval protein has the same amino acid sequence as the pupal protein. The sequence is characterized by a high content of alanine, proline, valine, and tyrosine and the complete absence of acidic amino acid residues, the sulphur containing amino acids and tryptophan. The sequence is further characterized by a high frequency of repeated sequence motifs, among which the Ala-Ala-Pro-Ala motif is the most abundant, but also longer sequence motifs are repeated. The sequence shows striking resemblance to sequences of proteins isolated from pharate locust cuticle.

Amino Acid Sequence↗

PubMed1995

Insect cuticular proteins.

Insect cuticles are composite structural materials with mechanical properties optimal for their biological functions. The bulk properties of cuticles are to a large extent determined by the interactions between the various components, mainly the chitin filament system and the proteins. The various cuticular types show pronounced differences in mechanical properties, and it is suggested that these differences can be related to the properties of the individual proteins and to the degree of secondary stabilization (sclerotization). The amino acid sequences, which have been obtained for insect cuticular proteins either by direct sequencing of purified proteins or by deduction from corresponding DNA-sequences, are listed according to insect order and species. Extensive sequence similarity is observed among several cuticular proteins obtained from different insect orders. Other cuticular proteins are characterized by repeated occurrence of a few small motifs consisting mainly of hydrophobic residues. The latter group of proteins has so far only been reported from stiff cuticles. The possible relevance of the various motifs and repeats for protein interaction and the mechanical properties of cuticles is discussed.

Amino Acid Sequence↗

PubMed1995

Primary structure of proteins from the wing cuticle of the migratory locust, Locusta migratoria.

Wing cuticle from pharate adult locusts, Locusta migratoria, contains several prominent proteins which occur as minor components or are completely absent in other cuticular regions. Six of the wing-specific proteins have been purified and their amino acid sequences determined by combined use of mass spectrometry and automated Edman degradation. During the sequence determination very long sequence runs (90-121 residues) were necessary in order to establish the primary structure. All the wing-specific cuticular proteins from locusts contain the repeated short sequence motif -Ala-Ala-Pro-Ala/Val-, which is common for all hitherto sequenced cuticular proteins from pharate locusts. Several of the wing-specific proteins also possess an N-terminal region rich in glycine, tyrosine and leucine, characteristic for many locust cuticular proteins. Two of the analysed proteins have a conserved 61-residue sequence in common with a previously sequenced protein from locust wing cuticle and with two proteins from the pharate cuticle of adult Tenebrio molitor. Possible roles for the various sequence motifs are discussed.

Amino Acid Sequence↗