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Epistasis is the dependency of the effect of a mutation on the genetic background in which it occurs. Epistasis has been widely documented and implicated in the evolution of species barriers and the evolution of genetic architecture. Here we propose a simple model to formalize the idea that epistasis can also lead to co-evolutionary patterns in molecular evolution of interacting genes. This model epistasis is represented by the influence of one gene substitution on the fitness rank of the resident allele at another locus. We assume that increasing or decreasing fitness rank occur equally likely. In simulations we show that this form of epistasis leads to co-evolution in the sense that the length of an adaptive walk between interacting loci is highly correlated. This effect is caused by episodes of elevated rate of evolution in both loci simultaneously. We find that the influence of epistasis on these measures of co-evolutionary dynamics is relatively robust to the details of the model. The main factor influencing the correlation in evolutionary rates is the probability that a substitution will have an epistatic effect, but the strength of epistasis or the asymmetry of the initial fitness ranks of the alleles have only a minor effect. We suggest that covariance in rates of evolution among loci could be used to detect epistasis among loci.

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.

The Fmoc-based SPPS of H-Xaa-Asp(OBzl)-Yaa-Gly-NH(2) sequences results in side reactions yielding not only aspartimide peptides and piperidide derivatives, but also 1,4-diazepine-2,5-dione-peptides. Evidence is presented to show that the 1,4-diazepine-2,5-dione derivative is formed from the aspartimide peptide. The rate of this ring transformation depends primarily on the tendency to aspartimide and piperidide formation, which is influenced by the nature of the amino acid following the aspartic acid beta-benzyl ester (Xaa). However the bulkiness of the amino acid side chain preceeding the aspartic acid beta-benzyl ester (Yaa) is also important. Under certain conditions the 1,4-diazepine-2,5-dione peptide derivative may even be formed dominantly, which is a highly undesirable side reaction in peptide synthesis, but which provides a new way for the synthesis of diazepine peptide derivatives with targeted biological or pharmacological activity., ((c) 2007 European Peptide Society and John Wiley & Sons, Ltd.)

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.

Studies to test the noxious effect of mercury ions on wheat germination and seedling growth showed that germination rate, shoot length, and fresh weights varied as a function of their concentration in the treatment solutions. At the same concentration, the radioactive mercury proved to be more harmful to the living seeds and seedlings. The detoxification action of glutathione for both radioactive and non-radioactive mercury was also followed. After a seven-day period of germination in the presence of the investigated compounds, the wheat plantlets were cut from the seeds, and their height, weight, and residual radioactivity were measured. The shoot length decreased from 8.1 (blank) to 4.6 cm (non-radioactive mercury) or even to 2.5 cm ((203)Hg), while glutathione had both an anti-toxic and an anti-radiotoxic effect (6.4 and 6.0 cm, respectively). The root weight of the lot decreased from 1.7 to 0.7 g and 0.4 g, respectively, while glutathione showed a healing action (1.5 and 1.7 g). The radioactive ions accumulated especially in roots (35.5 %), and less in shoots (11.2 %). Results were statistically validated.

Shotgun proteomics was used to study the steady phosphorylation state of NADH:ubiquinone oxidoreductase (complex I) subunits from bovine heart mitochondria. A total tryptic digestion of enzymatically active complex I was performed, and the resulting peptide mixture was subjected to phosphopeptide enrichment by the use of titanium dioxide (TiO2). The phosphopeptide-enriched fraction was separated and analyzed with nanoscale reverse-phase HPLC-ESI-MS/MS in single information-dependent acquisition. Hence two phosphorylated complex I subunits were detected: 42 kDa and B14.5a. Phosphorylation of 42-kDa subunit at Ser-59 has already been determined with fluorescent phosphoprotein-specific gel staining and mass spectrometry (Schilling, B., Aggeler, R., Schulenberg, B., Murray, J., Row, R. H., Capaldi, R. A., and Gibson, B. W. (2005) Mass spectrometric identification of novel phosphorylation site in subunit NDUFA10 of bovine mitochondrial complex I. FEBS Lett. 579, 2485-2490). In our work, this finding was confirmed using a non-gel-based approach. In addition, we report novel phosphorylation on B14.5a nuclear encoded subunit. We demonstrated evidence of the phosphorylation site at Ser-95 residue by collision-induced dissociation experiments on three different molecular ions of two tryptic phosphopeptides of B14.5a.

In this work, copper-binding of the tetraglycine peptide (Gly-Gly-Gly-Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M+2Cu-3H](+) and [4M+3Cu-5H](+). At 1:1 peptide-copper(II) ion ratio, the singly charged [3M+2Cu-3H](+) oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomer-ization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the -mechanism of metal ion binding and metal ion-induced oligomerization of peptides.

Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of acetamidomethyl (Acm) and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds leads up to the appropriate compound in the case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the nonnative disulfide isomer.

The 6-coordinate dioximatomanganese(II) complex [Mn(HL)(CH3OH)]+ (2, where H2L is [HON=C(CH3)C(CH3)=NCH2CH2]2NH), formed by instant solvolysis of [Mn2(HL)2](BPh4)2 (1) in methanol, accelerates the triethylamine (TEA)-catalyzed oxidation of 3,5-di-tert-butylcatechol (H2dtbc) by O2 to the corresponding o-benzoquinone. Significantly, 2 alone has no catalytic effect. The observed rate increase can be explained by the interaction of 2 with the hydroperoxo intermediate HdtbcO2- formed from Hdtbc- and O2 in the TEA-catalyzed oxidation. The kinetics of the TEA-catalyzed and Mn-enhanced reaction has been studied by gas-volumetric monitoring of the amount of O2 consumed. The initial rate of O2 uptake (V(in)) shows a first-order dependence on the concentration of 2 and O2 and saturation kinetics with respect to both H2dtbc and TEA. The observed kinetic behavior is consistent with parallel TEA-catalyzed and Mn-enhanced oxidation paths. The 3,5-di-tert-butylsemiquinone anion radical is an intermediate detectable by electron spin resonance (ESR) spectroscopy. The dimeric catalyst precursor has been characterized by X-ray diffraction and electrospray ionization mass spectrometry and the monomeric catalyst by ESR spectroscopy.

Here we propose shotgun proteomics as an alternative method to gel-based bottom-up proteomic platform for the structural characterization of mitochondrial NADH:ubiquinone oxidoreductase (complex I). The approach is based on simultaneous identification of subunits after global digestion of the intact complex. Resulting mixture of tryptic peptides is purified, concentrated, separated and online analyzed using nano-scale reverse-phase nano-ESI-MS/MS in a single information dependent acquisition mode. The usefulness of the method is demonstrated in our work on the well described model system of complex I from bovine heart mitochondria. The shotgun method led to the identification and partial sequence characterization of 42 subunits representing more than 95% coverage of the complex. In particular, almost all nuclear (except MLRQ) and 5 mitochondria DNA encoded subunits (except ND4L and ND6) were identified. Furthermore, it was possible to identify 30 co-purified proteins of the inner mitochondrial membrane structurally not belonging to complex I. The method's efficiency is shown by comparing it to two classical 1 D gel-based strategies. Shotgun proteomics is less laborious, significantly faster and requires less sample material with minimal treatment, facilitating the screening for post-translational modifications and quantitative and qualitative differences of complex I subunits in genetic disorders.

Cranial placodes are specialized regions of the ectoderm, which give rise to various sensory ganglia and contribute to the pituitary gland and sensory organs of the vertebrate head. They include the adenohypophyseal, olfactory, lens, trigeminal, and profundal placodes, a series of epibranchial placodes, an otic placode, and a series of lateral line placodes. After a long period of neglect, recent years have seen a resurgence of interest in placode induction and specification. There is increasing evidence that all placodes despite their different developmental fates originate from a common panplacodal primordium around the neural plate. This common primordium is defined by the expression of transcription factors of the Six1/2, Six4/5, and Eya families, which later continue to be expressed in all placodes and appear to promote generic placodal properties such as proliferation, the capacity for morphogenetic movements, and neuronal differentiation. A large number of other transcription factors are expressed in subdomains of the panplacodal primordium and appear to contribute to the specification of particular subsets of placodes. This review first provides a brief overview of different cranial placodes and then synthesizes evidence for the common origin of all placodes from a panplacodal primordium. The role of various transcription factors for the development of the different placodes is addressed next, and it is discussed how individual placodes may be specified and compartmentalized within the panplacodal primordium. Finally, tissues and signals involved in placode induction are summarized with a special focus on induction of the panplacodal primordium itself (generic placode induction) and its relation to neural induction and neural crest induction. Integrating current data, new models of generic placode induction and of combinatorial placode specification are presented.

The synthesis and chemotactic properties of a new class of branched oligopeptide-based conjugates are described. Tetratuftsin derivatives containing chemotactic formyl tripeptides (For-MLF, For-NleLF or For-MMM) in branches were prepared by stepwise solid-phase peptide synthesis. The influence of the composition and ionic charge of the carrier-branched oligopeptide on the chemotactic behaviour of the conjugate was studied in Tetrahymena pyriformis. Conjugates with methotrexate (Mtx) as a drug component was also prepared. For this, a GFLGC spacer, cleavable by cathepsin B, was used. The spacer with N-terminal methotrexate was coupled to the chloroacetylated chemotactic carrier molecule by thioether bond formation. The chemotactic activity and cytotoxity of Mtx conjugates were also studied., (Copyright (c) 2005 European Peptide Society and John Wiley & Sons, Ltd.)

Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.

The effect of matrix composition on signal suppression caused by a dominant compound under MALDI ionization was studied using the combinatorial TQTXT pentapeptide library as a model system. The peptide library is composed of 19 components with all proteinogenic amino acids except cysteine in position X. From these compounds, only the Arg peptide (TQTRT) was detected with sufficient intensity in the MALDI-TOF mass spectrum under typical MALDI conditions (CCA matrix). The analysis of a set of compounds utilized as different matrix components, additives and a cationizing agent revealed that the composition of the matrix is a critical point in signal suppression. Highly improved ion yields were achieved by using a CCA/DHB mixture as a matrix. The addition of K(+) as a cationizing agent to the CCA matrix resulted in MALDI-TOF mass spectra with relative ion intensities very similar to those obtained by electrospray ionization.

A new anionic surfactant (RapiGest SF) was successfully used for site-specific analysis of glycosylation in human alpha-1-acid glycoprotein (AGP). By means of this analytical approach combined with capillary HPLC-mass spectrometry (and tandem mass spectrometry), the N-linked glycosylation pattern of AGP was explored. On the basis of mass matching and MS/MS experiments ca 80 different AGP-derived glycopeptides were identified. Glycosylation shows a markedly different pattern for the various glycosylation sites. At sites I and II, triantennary complex-type oligosaccharides predominate and at sites III, IV and V, tetra-antennary complex-type oligosaccharides predominate. Sites IV and V show the presence of additional N-acetyl lactosamine (Gal-GlcNAc) units (even higher degree of branching and/or longer antennae are also present)., (Copyright 2005 John Wiley & Sons, Ltd)

Stability and structure of homo- and heterochiral protonated serine (Ser) dimers were investigated by a combination of mass spectrometry and ab initio quantum chemical calculations. This established that the energy difference between the most stable homo- and heterochiral forms is very small: tandem mass spectrometry with Cooks' kinetic method yielded a negligible difference in Gibbs free energy (0.2+/-0.2 kJ mol(-1)). The various isomeric forms of (Ser)2 H+ and their energetics were determined by extensive electronic-structure calculations, which yielded homo- and heterochiral forms of the isomers with distinctly different relative energies. The most stable homochiral isomer is stabilized by two hydrogen bonds and is far more stable than any other homochiral isomer. The most stable heterochiral isomer has completely different features, and it is characterized by a salt-bridge structure. This clearly shows that salt-bridge structures do exist in the gas phase even in comparatively small molecules and in the absence of particularly basic or acidic functional groups.

After primary neurogenesis in the Xenopus laevis embryo, a massive new surge of neurogenesis and related neurogenic and proneural gene expression occurs in the spinal cord at the beginning of the larval period (starting at Stage 46), which corresponds to well-documented secondary neurogenesis in larval zebrafish central nervous system development. Here, we document related neural proliferation and gene expression patterns in the brain of Xenopus, in various embryonic and larval stages, showing the distribution of proliferative cells (immunostaining of cells containing the proliferating cell nuclear antigen; the auxiliary protein of DNA polymerase delta; PCNA), and the activity of some critical genes expressed during neurogenesis (i.e., Delta-1, Neurogenin-related-1, NeuroD). This study reveals that the early larval stage in Xenopus (Stage 48) displays patterns of proliferation (PCNA), as well as of neurogenic (Delta-1) and proneural (Ngnr-1; NeuroD) gene expression that are qualitatively almost identical to those seen in the 3-day postembryonic zebrafish or the 12.5/13.5-day embryonic mouse. Furthermore, a comparable bauplan of early proliferation zones (including their neuromeric organization) as described in the postembryonic zebrafish apparently exists in tetrapods (Xenopus). Altogether, the data presented suggest a common brain bauplan on the level of early proliferation patterns and neurogenic/proneural gene activity in anamniotes, if not vertebrates., ((c) 2005 Wiley-Liss, Inc.)

Ectodermal placodes comprise the adenohypophyseal, olfactory, lens, profundal, trigeminal, otic, lateral line, and epibranchial placodes. The first part of this review presents a brief overview of placode development. Placodes give rise to a variety of cell types and contribute to many sensory organs and ganglia of the vertebrate head. While different placodes differ with respect to location and derivative cell types, all appear to originate from a common panplacodal primordium, induced at the anterior neural plate border by a combination of mesodermal and neural signals and defined by the expression of Six1, Six4, and Eya genes. Evidence from mouse and zebrafish mutants suggests that these genes promote generic placodal properties such as cell proliferation, cell shape changes, and specification of neurons. The common developmental origin of placodes suggests that all placodes may have evolved in several steps from a common precursor. The second part of this review summarizes our current knowledge of placode evolution. Although placodes (like neural crest cells) have been proposed to be evolutionary novelties of vertebrates, recent studies in ascidians and amphioxus have proposed that some placodes originated earlier in the chordate lineage. However, while the origin of several cellular and molecular components of placodes (e.g., regionalized expression domains of transcription factors and some neuronal or neurosecretory cell types) clearly predates the origin of vertebrates, there is presently little evidence that these components are integrated into placodes in protochordates. A scenario is presented according to which all placodes evolved from an adenohypophyseal-olfactory protoplacode, which may have originated in the vertebrate ancestor from the anlage of a rostral neurosecretory organ (surviving as Hatschek's pit in present-day amphioxus)., (Copyright 2005 Wiley-Liss, Inc.)

The association properties of natural and non-natural amino acids were studied in detail using electrospray ionization mass spectrometry. The results show a highly diverse cluster formation behavior of amino acids. There are differences regarding the degree of clustering (average cluster size), the presence or absence of one or several 'magic' clusters of special stability and the influence of chirality on cluster stability. Cluster formation does not show a good correlation with simple physico-chemical properties (such as solubility), indicating that it is a specific process and not only a simple aggregation during evaporation/ionization. A systematic study of cluster formation of serine derivatives reveals that all functional groups play a prominent role in the binding of the magic octamer. The results support the idea of the zwitterionic character of the octamer. Electrospray ionization of the side-chain acetylated serine shows the formation of a very stable tetramer with a strong preference for homochirality. The results suggest that Ser8 is made up of two tetramer subunits, held together by hydrogen bonds of the side-chain.

A simple and novel approach was developed to detect non-covalent interactions. It is based on combination of solid-phase affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). One of the interacting molecules is bound to magnetic beads and is incubated with the target molecules in solution. The complex bound on the solid support is removed from the solution and transferred for MALDI analysis. Mass spectrometry is used only to detect the target compound, which is far more straightforward than detecting the intact non-covalent complex. To demonstrate the applicability of the method, an AT-rich oligonucleotide (5'-CCCCCAATTCCCCC-3') and its complementary biotinylated sequence (5'-biotin-GGGGGAATTGGGGG-3') were hybridized and immobilized to paramagnetic particles by streptavidin-biotin interaction. The immobilized duplex oligonucleotide was reacted with minor groove binding drugs, Netropsin, Distamycin A, Hoechst 33258 and 4',6-diamidino-2-phenylindole. The resulting DNA-drug complex bound to the particles was separated and analyzed by linear MALDI-TOFMS after washing. Drugs were selectively detected in the spectra. Relative binding strengths were also estimated using competitive complexation., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.

We analyzed the spatiotemporal pattern of expression of 15 transcription factors (Six1, Six4, Eya1, Sox3, Sox2, Pax6, Pax3, Pax2, Pax8, Dlx3, Msx1, FoxI1c, Tbx2, Tbx3, Xiro1) during placode development in Xenopus laevis from neural plate to late tail bud stages. Out of all genes investigated, only the expression of Eya1, Six1, and Six4 is maintained in all types of placode (except the lens) throughout embryonic development, suggesting that they may promote generic placodal properties and that their crescent-shaped expression domain surrounding the neural plate defines a panplacodal primordium from which all types of placode originate. Double-labeling procedures were employed to reveal the precise position of this panplacodal primordium relative to neural plate, neural crest, and other placodal markers. Already at neural plate stages, the panplacodal primordium is subdivided into several subregions defined by particular combinations of transcription factors allowing us to identify the approximate regions of origin of various types of placode. Whereas some types of placode were already prefigured by molecularly distinct areas at neural plate stages, the epibranchial, otic, and lateral line placodes arise from a common posterior placodal area (characterized by Pax8 and Pax2 expression) and acquire differential molecular signatures only after neural tube closure. Our findings argue for a multistep mechanism of placode induction, support a combinatorial model of placode specification, and suggest that different placodes evolved from a common placodal primordium by successive recruitment of new inducers and target genes.

A simple method capable of generating and investigating various solvent clusters and solvated ions was developed. The technique opens a door to studying these complexes on commercially available instruments. Formation of the desired solvated ion in the gas phase was achieved by introducing the appropriate volatile solvent vapour into the curtain gas stream. Capabilities of the technique are illustrated by generating alkali, alkaline earth and transition metal cations solvated by various volatile compounds such as water, methanol and acetonitrile. Depending on the ligands and on the experimental conditions, clusters of 2-100 molecules may be observed. Isotope labelling suggests that these are formed by a re-solvation process in the curtain gas region., (Copyright 2003 John Wiley & Sons, Ltd.)

The recently discovered homochiral serine octamer has been a focus of interest because of its possible implications for the origin of homochirality in living systems. Electrospray ionization (ESI) and sonic spray ionization (SSI) tandem mass spectrometry have been used to generate this unusually stable magic number cluster. Several structures have been suggested for the serine octamer, based on tandem mass spectrometry, ion mobility measurements, and quantum mechanical calculations. We now report experimental hydrogen/deuterium (H/D) exchange data, which demonstrate the existence of two different structures for the serine octamer. These forms undergo exchange at significantly different rates. One form may correspond to solution-phase assembled clusters and the other to octamers formed during the ionization process. Experiments done at higher resolution confirm that the experimental observations made here apply to the serine octamer without interference from metaclusters, namely, higher order clusters (Ser(16) + 2H)(+2), etc., the (12)C isotopes of which have mass-to-charge ratios identical to the protonated octamers. H/D exchange of racemic serine shows predominantly the extensively exchanged ion population, as well as providing evidence that racemic serine generates abundant metaclusters. The evidence presented here shows that one type of serine octamer is responsible for the strong chiral effects associated with the formation of these magic number clusters. These slowly exchanging more fragile clusters are the octamers that might have played a role in homochirogenesis.

Two cyclic peptides with a thioether bond have been synthesised corresponding to the 9-22 (9LKMADPNRFRGKDL(22)) sequence of glycoprotein D (gD-1) of Herpes simplex virus. The role of the secondary structure in protein-specific monoclonal antibody recognition was investigated. The sequence selected for this study comprises a strongly antigenic site adopting a beta-turn at residues 14Pro-(15)Asn. Thioether bond was formed between the free thiol group of cysteine or homocysteine inserted in position 11 and the chloroacetylated side chain of lysine in position 18. We report here the preparation of cyclic peptides containing Cys or Hcy in position 11, differing only in one methylene group. The linear precursor peptides were synthesised by Boc/Bzl strategy on MBHA resin, and the cyclisation was carried out in alkaline solution. The secondary structure of the peptides was studied by CD, FT-IR and NMR spectroscopy. The CD and FT-IR data have revealed fundamental changes in the solution conformation of the two compounds. The CH(2) group difference significantly resulted in the altered turn structure at the 12Ala and 13Asp as identified by NMR spectroscopy. The antibody binding properties of the cyclopeptides studied by gD-specific monoclonal antibody (A16) in direct and competition enzyme-linked immunosorbent assay (ELISA) were also not the same. We found that peptide LK[HcyADPNRFK]GKDL exhibited higher affinity to Mab A16 than peptide LK[CADPNRFK]GKDL, however, their reactivity was significantly lower compared to the linear ones. Our results clearly show the importance of secondary structure in an antibody binding and demonstrate that even a slight modification of the primary structure dramatically could influence the immune recognition of the synthetic antigens.

Recently, a novel type of neurogenic placode was described in anurans. These hypobranchial placodes were recognized as ectodermal thickenings situated ventral to the second and third pharyngeal pouch that give rise to neurons of unknown fate. Here, the development of hypobranchial placodes in Xenopus laevis is described in more detail using in situ hybridization and immunohistochemistry for various placodal ( Six1, Eya1) and neurogenic ( NGNR-1, NeuroD, Delta-1, Hu, acetylated tubulin) markers. Moreover, the fate of hypobranchial placodes was determined by analyzing tadpoles that had received orthotopic grafts of ventral branchial arch ectoderm at embryonic stages from donor embryos injected with the lineage tracer green fluorescent protein. The neurogenic epibranchial and hypobranchial placodes are shown to develop in certain subregions of a broader branchial placodal area as defined by Six1 and Eya1 expression, viz., adjacent to the dorsal and ventral tip of the pharyngeal pouches, respectively. Grafting experiments show that each of the two hypobranchial placodes gives rise to a small and previously undescribed hypobranchial ganglion (identified by its immunoreactivity for the neuron-specific Hu protein) of unknown function located in the ventral branchial arch region. No contributions of hypobranchial placodes to any other ganglia (including cardiac ganglia and the ganglia of branchiomeric nerves located dorsal to pharyngeal pouches) were found.

A novel ion source based on the principle of sonic spray ionization has been built and used to optimize mass spectrometric conditions for generating amino acid clusters. The ion source employs a simple pneumatic spray operated at extremely high nebulizing gas flow rates. Several factors that affect the performance of the cluster source are identified, and information from these observations provides insights into the mechanisms of gas phase ion formation. Serine is used as a model system in optimizing instrumental and sample parameters to maximize cluster ion formation. The sonic spray results for this oligomer compare favorably with electrospray data, showing an order of magnitude better signal intensity and excellent signal-to-noise ratios. The performance of the system for the protonated serine octamer includes a limit of detection of 10 nM and a linear dynamic range of 4 orders of magnitude. Ion formation was observed to go into saturation above 1 mM. This result and data on pH, electrolyte concentration, and solvent composition are interpreted as supporting a charge residue model of sonic spray ionization. Other amino acids can be substituted for serine in the octamer, with a strong chiral preference in favor of homochiral cluster formation in the cases of threonine and cysteine. These amino acids show a preference for substitution of more than two serine molecules. Phenylalanine, asparagine, tryptophan, and tyrosine also substitute into the serine octamer; however, the process yields only two incorporations and only small chiral effects.

Previous studies have shown that spinal cord development in direct developing frogs of the genus Eleutherodactylus, which have evolutionarily lost the tadpole stage, differs from that in biphasically developing anurans (with the larval and the adult stage separated by metamorphosis). The present study of spinal cord development in Eleutherodactylus coqui provides additional information about neurogenesis, neuronal differentiation and growth analyzed by immunostaining for proliferating cell nuclear antigen (PCNA), in situ hybridization for NeuroD, and morphometric measurements in various developmental stages. Furthermore, spinal cord development in the frogs Discoglossus pictus, Xenopus laevis, and Physalaemus pustulosus, which belong to different anuran families but all exhibit biphasic development, was similarly analyzed. This comparative analysis allows inference of the ancestral anuran pattern of spinal cord development and how it has been modified during the evolution of Eleutherodactylus. All biphasically developing frogs analyzed share a similar pattern of spinal cord development, suggesting that this is ancestral for anurans: after neural tube closure, levels of proliferation and neurogenesis in the spinal cord were low throughout embryogenesis until they were upregulated drastically at early larval stages followed by development of the lateral motor columns. In contrast, no such quiescent embryonic period exists in E. coqui, where rapid growth, high levels of proliferation and neurogenesis, and early formation of lateral motor columns occur shortly after neural tube closure, while other parts of the central nervous system develop more slowly. Thus, spinal cord development has been accelerated during the evolution of Eleutherodactylus relative to the development of other parts of the central nervous system, probably related to the precocious development of limbs in this lineage.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to detect an immune complex formed between beta-lactoglobulin and polyclonal anti-beta-lactoglobulin antibody in the gas phase. The most important experimental parameters to detect such a specific antibody-antigen complex by MALDI were the use of solutions at near-neutral pH and of sinapinic acid matrix prepared by the dried-droplet method. Under such conditions, predominantly one but also two molecules of antigen protein were complexed by the antibody. Specific formation of the antibody-antigen complex was confirmed by performing competitive reactions. Addition of antibody to a 1:1 mixture of beta-lactoglobulin and one control protein resulted not only in the appearance of the expected antibody-antigen complex, but also in a strong decrease in the free beta-lactoglobulin signal, while the abundance of the control protein was not influenced., (Copyright 2003 John Wiley & Sons, Ltd.)

Three phases of neurogenesis can be recognized during Xenopus spinal cord development. An early peak during gastrulation/neurulation is followed by a phase of low level neurogenesis throughout the remaining embryonic stages and a later peak at early larval stages. We show here that several genes known to be essential for early neurogenesis (X-NGNR-1, XNeuroD, XMyT1, X-Delta-1) are also expressed during later phases of neurogenesis in the spinal cord, suggesting that they are involved in regulating spinal neurogenesis at later stages. However, additional neuronal determination genes may be important during larval stages, because X-NGNR-1 shows only scant expression in the spinal cord during larval stages. Thyroid hormone treatment of early larvae promotes neurogenesis in the spinal cord, where thyroid hormone receptor xTRalpha is expressed from early larval stages onward and results in precocious up-regulation of XNeuroD, XMyT1, and N-Tubulin expression. Similarly, thyroid hormone treatments of Xenopus embryos, which were coinjected with xTRalpha and the retinoid X receptor xRXRalpha, repeatedly resulted in increased numbers of neurons, whereas unliganded receptors repressed neurogenesis. Our findings show that thyroid hormones are sufficient to up-regulate neurogenesis in the Xenopus spinal cord., (Copyright 2002 Wiley-Liss, Inc.)

The amphibian lateral line system develops from a series of lateral line placodes. The different phases of development from early induction, to pattern formation, differentiation, morphogenesis, and metamorphic fate were summarized in the first part of this review (Schlosser, 2002a). Here, a survey of the diversity of lateral line systems in amphibians is presented indicating that most phases of lateral line development have been subject to evolutionary changes. Several trends suggest important roles for both adaptive changes and internal constraints in amphibian lateral line evolution. Many of these trends involved the coordinated modification of different derivatives of lateral line placodes suggesting that these placodes are not only autonomous developmental modules, but also units of evolutionary variation that tend to be modified in a coherent and largely context-independent fashion.

Lateral line placodes are specialized regions of the ectoderm that give rise to the receptor organs of the lateral line system as well as to the sensory neurons innervating them. The development of lateral line placodes has been studied in amphibians since the early 1900s. This paper reviews these older studies and tries to integrate them with more recent findings. Lateral line placodes are probably induced in a multistep process from a panplacodal area surrounding the neural plate. The time schedule of these inductive processes has begun to be unravelled, but little is known yet about their molecular basis. Subsequent pattern formation, morphogenesis and differentiation of lateral line placodes proceeds in most respects relatively autonomously: Onset and polarity of migration of lateral line primordia, the type, spacing, size and number of receptor organs formed, as well as the patterned differentiation of different cell types occur normally even in ectopic locations. Only the pathways for migration of lateral line primordia depend on external cues. Thus, lateral line placodes act as integrated and relatively context-insensitive developmental modules.

Mechanisms that mediate limb development are regarded as highly conserved among vertebrates, especially tetrapods. Yet, this assumption is based on the study of relatively few species, and virtually none of those that display any of a large number of specialized life-history or reproductive modes, which might be expected to affect developmental pattern or process. Direct development is an alternative life history found in many anuran amphibians. Many adult features that form after hatching in metamorphic frogs, such as limbs, appear during embryogenesis in direct-developing species. Limb development in the direct-developing frog Eleutherodactylus coqui presents a mosaic of apparently conserved and novel features. The former include the basic sequence and pattern of limb chondrogenesis, which are typical of anurans generally and appear largely unaffected by the gross shift in developmental timing; expression of Distal-less protein (Dlx) in the distal ectoderm; expression of the gene Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA); and the ability of the ZPA to induce supernumerary digits when transplanted to the anterior region of an early host limb bud. Novel features include the absence of a morphologically distinct apical ectodermal ridge, the ability of the limb to continue distal outgrowth and differentiation following removal of the distal ectoderm, and earlier cessation of the inductive ability of the ZPA. Attempts to represent tetrapod limb development as a developmental "module" must allow for this kind of evolutionary variation among species., (Copyright 2001 Wiley-Liss, Inc.)

The comparison of developmental sequences among species is notoriously difficult. Here, heterochrony plots are introduced as a new graphic method to detect temporal shifts in the development of characters in pair-wise species comparisons. Plotting the timing of character development in one species against the timing of character development in another species allows us to compare a principally unlimited number of characters simultaneously and can detect whether suites of characters are dissociated from one another or not. Such heterochrony plots can be embedded into a comparative phylogenetic analysis in order to establish whether observed patterns of character codissociation are indeed due to their dissociated coevolution. Comparative phylogenetic analysis may also reveal multiple independent events of dissociated coevolution of the same suite of characters in a certain lineage, suggesting that the characters of this suite reciprocally constrain their evolutionary modifiability, thereby forming a unit of evolution. This ability to identify units of evolution is a prerequisite for assessing the validity of recently proposed scenarios, suggesting that modules of development and/or function tend to act as units of evolution. Starting from a detailed heterochrony plot comparing development in the direct developing frog Eleutherodactylus coqui and in the biphasically developing frog Discoglossus pictus, this comparative approach is illustrated focusing on the evolution of development of limbs, the nervous system and the pharyngeal arches in amphibians., (Copyright 2001 Wiley-Liss, Inc.)

In order to determine the time window for induction of lateral line placodes in the axolotl, we performed two series of heterotopic and isochronic transplantations from pigmented to albino embryos at different stages of embryogenesis and assessed the distribution of pigmented neuromasts in the hosts at later stages. First, ectoderm from the prospective placodal region was transplanted to the belly between early neurula and mid tailbud stages (stages 13-27). Whereas grafts from early neurulae typically differentiated only into epidermis, grafts from late neural fold stages on reliably resulted in differentiation of ectopic pigmented neuromasts. Second, belly ectoderm was transplanted to the prospective placodal region between early neurula and tailbud stages (stages 13-35). Normal lateral lines containing pigmented neuromasts formed in most embryos when grafts were performed prior to early tailbud stages (stage 24) but not when they were performed later. Our findings indicate that lateral line placodes, from which neuromasts originate, are already determined at late neural fold stages (first series of grafts) but are inducible until early tailbud stages (second series of grafts). A further series of heterochronic transplantations demonstrated that the decline of inducibility at mid tailbud stages is mainly due to the loss of ectodermal competence., (Copyright 2001 Academic Press.)

Objective and Design: Therapeutic use and safety of a pumpkin seed extract were tested in a multicentric clinical trial., Patients and Methods: 2,245 patients suffering from benign prostate hyperplasia (stage I to II according to Alken) received for 12 weeks 1-2 capsules Prosta Fink Forte per day. Urinary symptoms were recorded by the International Prostate Symptom Score according to the American Urological Association (I-PSS), the influence on quality of life has been recorded by a quality of life questionnaire (LQ Index)., Results: The I-PSS decreased by 41.4%, life quality improved by 46. 1% during therapy. More than 96% of the patients reported no undesired side effects under the treatment with Prosta Fink Forte., Conclusion: Prosta Fink Forte facilitates an efficient improvement of BPH symptoms, especially in early stages., (Copyright 2000 S. Karger GmbH, Freiburg)

The development of neurogenic placodes in Xenopus laevis from the time of neural fold closure to larval stages is described. Placodes were reconstructed from camera lucida drawings of serial sections, and the spatiotemporal pattern of placodal neurogenesis was analyzed using in situ hybridization for the genes X-NGNR-1, XNeuroD, X-MyT1, and X-Delta-1, all of which have been implicated in the regulation of neurogenesis. Olfactory, profundal, and trigeminal placodes, a series of dorsolateral placodes (otic placode and five lateral line placodes), a series of epibranchial placodes, and two hypobranchial placodes were identified. Earlier claims that all placodes in anurans develop from a common primordium could not be confirmed. Profundal and trigeminal placodes, however, are partially fused, and all lateral line placodes arise from a common precursor. Epibranchial and hypobranchial placodes develop ventral to other placodes and dorsal and ventral to the pharyngeal pouches, respectively. Hypobranchial placodes give rise to neurons that become intimately associated with the developing heart. All neurogenic placodes strongly express the neuronal differentiation gene XNeuroD. The neuronal determination gene X-NGNR-1, however, is expressed strongly in only some placodes and not in dorsolateral placodes, indicating that neurogenesis in the latter relies on other determination genes. X-Delta-1 is expressed not only in the neurogenic parts of the placodes but also in the primordia of the lateral lines. This suggests that Delta-Notch-mediated lateral inhibition may be involved not only in placodal neurogenesis, but also in the patterning of lateral line neuromasts.

In the direct-developing frog Eleutherodactylus coqui neuromasts and ganglia of the lateral line system never develop. We show here that this absence of the lateral line system, which is evolutionarily derived in anurans, is due to very early changes in development. Ectodermal thickenings, which are typical of lateral line placodes, and from which neuromasts and ganglion cells of the lateral line originate, never form in E. coqui, although other neurogenic placodes are present. Moreover, although NeuroD is expressed in the lateral line placodes of Xenopus laevis, corresponding expression sites are lacking in E. coqui. Heterospecific transplantation experiments show that axolotl ectoderm can be induced to form lateral line placodes after transplantation to E. coqui hosts but that E. coqui ectoderm does not form lateral line placodes on axolotl hosts. This suggests that the loss of the lateral line system in E. coqui is due to the specific loss of ectodermal competence to form lateral line placodes in response to inductive signals. Our results (1) indicate that the competence for lateral line placode formation is distinct and dissociable from the competence to form other neurogenic placodes and (2) support the idea that the lateral line system acts as a module in development and evolution., (Copyright 1999 Academic Press.)

The loss of a free-living larval stage during the evolution of directly developing frogs of the genus Eleutherodactylus resulted in dramatic alterations in ontogeny. Immunostaining for proliferating cell nuclear antigen reveals that in the directly developing frog Eleutherodactylus coqui pervasive cell proliferation occurs throughout the retina even after the plexiform layers have formed. In striking contrast to biphasically developing frogs (e.g. Discoglossus pictus or Xenopus laevis), in E. coqui proliferation becomes restricted to the ciliary margin only after the eye has reached the size typical of a postmetamorphic froglet and after its laminar structure has developed. As a consequence, the retina of E. coqui develops rapidly without recapitulating larva-typical stages. Our results suggest that dissociation of cell proliferation and differentiation can lead to the abbreviation of ontogenies during evolution.

We use whole-mount immunohistochemistry to describe the pattern of development of cranial nerves and muscles in the direct-developing leptodactylid frog Eleutherodactylus coqui. Comparison with nerve development in the biphasically developing frogs Physalaemus pustulosus (Leptodactylidae) and Discoglossus pictus (Discoglossidae; described in a companion paper) allows us to infer the ancestral leptodactylid ontogenetic pattern and the extent to which it has been modified during the evolution of direct development in Eleutherodactylus. While early embryonic development of cranial nerves and muscles is remarkably conserved in E. coqui, most transitory embryonic and larval characters (e.g., occipital and spinal myotomes together with their innervation, the distorted course of trigeminal and facial nerves, ventral branchial arch muscles, a subset of branchial-nerve rami and the lateral-line system) never develop. However, a few larva-typical characters are recapitulated, including Rohon-Beard cells and an anastomosis between the vagal and hypoglossal nerve. In addition to the abbreviation of ontogeny by loss of larva-specific traits, dramatic dissociations and heterochronic shifts of characters can be observed in E. coqui. The onset of development of limb and trunk innervation has been pre-displaced to early embryonic stage. Moreover, the reorientation of cranial muscles and nerves corresponding to late metamorphic events in biphasically developing anurans occurs relatively much earlier and is less pronounced in E. coqui resulting in an extreme condensation of ontogeny.

The gross anatomical development of the peripheral nervous system (PNS) during embryogenesis and metamorphosis in the frog Discoglossus pictus is described based on whole-mount immunostaining for nerves and muscles. In the head, neurite outgrowth starts with the mandibular ramus of the trigeminal nerve at the tailbud stage. Cranial muscles are innervated as soon as they differentiate, beginning at mid-embryonic stages. During late embryonic stages, the course of the trigeminal and facial nerves becomes greatly distorted and changes again drastically during metamorphosis accompanying the reorganization of the jaw muscles. Two occipital somites and nerves develop transitorily but degenerate at late embryonic stages. The hypoglossal nerve develops by fusion of the first and second spinal nerves and receives a transitory contribution of the third and fourth spinal nerve at embryonic stages. In the trunk, several classes of Rohon-Beard neurites could be identified at embryonic stages, one of which forms intersegmental sensory nerves that prefigure the course of the sensory rami of spinal nerves at later stages. We give detailed schedules of PNS and cranial muscle development which, in comparison with data on other frog species described in a companion paper, will serve as a basis to evaluate heterochronic shift during evolution of PNS development in frogs.

The subcutaneous implanted venous port device offers a safe central venous access and allows for ambulatory treatment. This case report describes a late manifestation of a pneumothorax after venous port implantation which, in fact, is a rare but significant complications. The lack of a correlation of the signs and symptoms with the interventional procedure may lead to inadequate or delayed treatment. The immediate postprocedural X-ray can be false negative when air leaks into the pleural cavity very slowly. Therefore both patients and referring physicians should be informed about the possibility of the late manifestation of a pneumothorax.

We studied the peripheral nervous system of early tadpoles of the frog Discoglossus pictus using whole-mount immunohistochemistry. Double-labeling of muscles and nerves allowed us to determine the innervation of all cranial muscles supplied by the trigeminal, facial, glossopharyngeal, vagal, and hypoglossal nerves. The gross anatomical pattern of visceral, cutaneous, and lateral-line innervation was also assessed. Most muscles of the visceral arches are exclusively supplied by posttrematic rami of the corresponding branchiomeric nerves, the only exceptions being some ventral muscles (intermandibular, interhyoid, and subarcual rectus muscles). In the mandibular arch, the pattern of motor ramules of the trigeminal nerve prefigures in a condensed form the adult pattern, but the muscles of the hyoid arch are innervated by ramules of the facial nerve in a pattern that differs from that of postmetamorphic frogs. With respect to the nerves of the branchial arches, pretrematic visceral rami, typical of other gnathostomes, are absent in D. pictus. Instead, we find a separate series of posttrematic profundal visceral rami. Pharyngeal rami of all branchial nerves contribute to Jacobson's anastomosis. We provide a detailed description of the lateral-line innervation and describe a new ramus of the middle lateral-line nerve (ramus suprabranchialis). We confirm the presence of a first spinal nerve and its contribution to the hypoglossal nerve in D. pictus tadpoles.

There are certain demands on central venous access devices in the ambulatory or long-term parenteral treatment of end-stage cancer-patients. The devices should offer both save and easy access, low related morbidity and acceptance of the patients. Ambulatory implantable venous ports fulfill all the these demands as own experiences with 135 implantations in cancer patients show: an overall complication rate of 6% which is - compared to other systems - very low, good acceptance of the patients and low costs. The venous ports can reduce duration of in-house treatment, bears a low morbidity and is the patients first choice. The port device is competitive and cost efficient alternative to conventional central venous access devices.