Microbiological contamination of raw materials for large-volume parenterals.

A wide range of raw materials used for the production of large-volume parenterals were tested for their content of bacteria, molds, yeasts, and endotoxins. All raw materials were relatively free of microorganisms, although some mannitol samples contained relatively high amounts of endotoxin. The low endotoxin content of the other raw materials did not require the use of depth filters to remove endotoxins to prepare pyrogen-free infusion fluids.     

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.     

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K(m) values of Neb-ODAIF and Neb-ODAIF(1-9) (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 microM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K(m/i)=90 microM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.     

Uncovering conserved patterns in bioactive peptides in Metazoa

Bioactive (neuro)peptides play critical roles in regulating most biological processes in animals. Peptides belonging to the same family are characterized by a typical sequence pattern that is conserved among the family's peptide members. Such a conserved pattern or motif usually corresponds to the functionally important part of the biologically active peptide. In this paper, all known bioactive (neuro)peptides annotated in Swiss-Prot and TrEMBL protein databases are collected, and the pattern searching program Pratt is used to search these unaligned peptide sequences for conserved patterns. The obtained patterns are then refined by combining the information on amino acids at important functional sites collected from the literature. All the identified patterns are further tested by scanning them against Swiss-Prot and TrEMBL protein databases. The diagnostic power of each pattern is validated by the fact that any annotated protein from Swiss-Prot and TrEMBL that contains one of the established patterns, is indeed a known (neuro)peptide precursor. We discovered 155 novel peptide patterns in addition to the 56 established ones in the PROSITE database. All the patterns cover 110 peptide families. Fifty-five of these families are not characterized by the PROSITE signatures, and 12 are also not identified by other existing motif databases, such as Pfam and SMART. Using the newly identified peptide signatures as a search tool, we predicted 95 hypothetical proteins as putative peptide precursors.     

Purification, isolation and search for possible functions of a phase-related 6080-Da peptide from the haemolymph of the desert locust, Schistocerca gregaria

Abstract. A novel 6-kDa peptide has been isolated from the haemolymph of the desert locust, Schistocerca gregaria (Forskal) (Orthoptera: Acrididae) , and fully identified. Its concentration is higher in crowd-reared (gregarious) animals than in isolated-reared (solitarious) ones. Its concentration decreases progressively from generation to generation with solitarization of gregarious locusts. The peptide is also present in freshly laid eggs. The concentration in eggs is higher in those from crowd-reared locusts. It is likely that the peptide is transferred from the female's haemolymph into the eggs because injection of the peptide into females before oviposition increases the amount of the 6-kDa peptide in the eggs. A two-step HPLC purification procedure for this peptide is described. It allowed several bioassays to test for a possible function. Although the peptide's concentration in the haemolymph is high (0.1 m m ), which suggests some physiological function, we have as yet not been able to identify a function. We hypothesize that the 6-kDa peptide may somehow play a role as a maternal factor in the determination of the phase-state of the offspring.     

Microbiological contamination of raw materials for large-volume parenterals

A wide range of raw materials used for the production of large-volume parenterals were tested for their content of bacteria, molds, yeasts, and endotoxins. All raw materials were relatively free of microorganisms, although some mannitol samples contained relatively high amounts of endotoxin. The low endotoxin content of the other raw materials did not require the use of depth filters to remove endotoxins to prepare pyrogen-free infusion fluids.     

Comparative peptidomics of Caenorhabditis elegans versus C. briggsae by LC-MALDI-TOF MS

Neuropeptides are important signaling molecules that function in cell-cell communication as neurotransmitters or hormones to orchestrate a wide variety of physiological conditions and behaviors. These endogenous peptides can be monitored by high throughput peptidomics technologies from virtually any tissue or organism. The neuropeptide complement of the soil nematode Caenorhabditis elegans has been characterized by on-line two-dimensional liquid chromatography and quadrupole time-of-flight tandem mass spectrometry (2D-nanoLC Q-TOF MS/MS). Here, we use an alternative peptidomics approach combining liquid chromatography (LC) with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to map the peptide content of C. elegans and another Caenorhabditis species, Caenorhabditis briggsae. This study allows a better annotation of neuropeptide-encoding genes from the C. briggsae genome and provides a promising basis for further evolutionary comparisons.     

In silico identification of new secretory peptide genes in Drosophila melanogaster

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.     

Peptidomics of the locust corpora allata: identification of novel pyrokinins (-FXPRLamides)

The peptidomes of the corpora allata of Locusta migratoria and Schistocerca gregaria were investigated by both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography quadrupole time-of-flight tandem mass spectrometry (nanoLC-Q-TOF MSMS). The pyrokinin (-FXPRLamide) family seems to be predominant. In addition to the known pyrokinins, we de novo sequenced four pyrokinins in L. migratoria and five in S. gregaria. In addition, one pyrokinin-like peptide (-PRLamide) was identified in S. gregaria. Besides the -(FX)PRLamides, FLRFamide-1, the allatostatins (A family) and numerous as yet unidentified peptides are also present in the corpora allata.