Direct sequence analysis of proteins by in-source fragmentation during delayed ion extraction.

Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix-assisted laser desorption/ionization-generated ion signals dominated by Cn fragmentation within the ion source of a linear time-of-flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.     

Biomass and feeding activity of phagotrophic mixotrophs in the northwestern Black Sea during the summer 1995

Biomass and activities of planktonicmicroorganisms (bacteria, nanoplankton andmicroplankton) were measured in the northwestern BlackSea during summer 1995. The method based on theuptake of fluorescently labeled prey was chosen todetermine the ingestion rate of bacteria andnanoplankton by phagotrophic microorganisms. Thismethod revealed the presence of mixotrophic organismssuch as ’plastid-retaining ciliates‘ in the wholecoastal area. Mixotrophic ciliates were dominated bymicro-sized forms and maximum biomasses were recorded inthe water masses characterised by low nutrientconcentrations but high food particle concentrations. Mixotrophic nanoflagellates were absentand mixotrophic dinoflagellates were observed at onestation only. Mixotrophic ciliates were shown to ingestpreferably bacteria while mixotrophic dinoflagellateswere grazing almost exclusively on nanoflagellates.Although the biomass of mixotrophic organisms weresignificantly lower than those of aplastidic protozoa,their feeding activity contributed to 14 and 24% ofthe ingestion of bacteria and nanoplankton, respectively.This is due to the high specificingestion rate of mixotrophic micro-sized ciliates anddinoflagellates, which were two and three times higher,respectively, than the specific ingestion rate ofbacteria and nanoplankton by aplastidic protozoa. Thissuggests a significant contribution of phagotrophicmixotrophs to the microbial network of thenorthwestern Black Sea. This revised version was published online in July 2006 with corrections to the Cover Date.     

On the production,elemental composition (C,N, P) and distribution of photosynthetic organic matter in the Southern Black Sea

Chemical oceanographic understanding of the southernBlack Sea has been improved by recent measurements ofthe optical transparency, phytoplankton biomass (interms of chlorophyll-a and particulate organic matter)and primary productivity. During the spring-autmunperiod of 1995–1996, light generally penetrated onlyinto the upper 15–40 m, with an attenuation coefficientvarying between 0.125 and 0.350 m^2122;1. The averagechlorophyll-a (Chl-a) concentrations for the euphoticzone ranged from 0.1 to 1.5 μg l^2122;1. Coherentsub-surface Chl-a maxima were formed near the base ofthe euphotic zone only in summer. Production rate variedbetween 247 and 1925 in the spring and between 405 and687 mgC m^2122;2 d^2122;1 in the summer-autumn period.The average POM concentrations in the euphotic zonevaried regionally and seasonally between 3.8 and28.6 μm for POC, 0.5 and 3.1 μm for PON and0.02 and 0.1 μm for PP. Atomic ratios of C/N, C/Pand N/P, derived from the regressions of POM data,ranged between 7.5 and 9.6, 109 and 165, and 11.2 and16.6, respectively. In the suboxic/anoxic interface,the elemental ratios change substantially due to anaccumulation of PP cohering to Fe and Mn oxides. Thechemocline boundaries and the distinct chemicalfeatures of the oxic/anoxic transition layer (the so-called suboxic zone) are all located at specificdensity surfaces; however, they exhibit remarkablespatial and temporal variations both in their positionand in their magnitude, which permit the definition of long-term changes in the biochemical properties of theBlack Sea upper layer. This revised version was published online in July 2006 with corrections to the Cover Date.     

The impact of the Scheldt input on the trace metal distribution in the Belgian coastal area (results of 1981–1983 and 1995–1996)

Offshore fluxes of Cu, Zn, Cd, Pb and Hg were calculated based onresidual flow patterns and salinity gradients along the Belgian coast. Theresidual flow lines along the Belgian coast are more or less parallel to thecoast except in the area where the north-easterly flowing watermass comingfrom the Channel encounters the south-westerly-oriented Scheldt outflow,forming a residual hydrodynamical front. From the steady-state salinitypattern, diffusion coefficients perpendicular to the residual flow werededuced; they ranged from 21 to 108 m² s^-1. Offshore fluxes of dissolved and particulate trace metals based on diffusiveand mixing processes are calculated. The steady state profiles of dissolvedmetals show a dilution effect in the coastal waters, reaching an almostconstant concentration in the marine watermass in the 1981–1983dataset. The ratios of the Scheldt input of trace metals to the totaldissolved offshore flux vary from 38 to 55% (1981–1983),depending on the kind of metal, and from 55 to 91% (1995–1996).The ratio of the Scheldt input to the dissolved metal flow parallel to thecoast, is in both periods (1981–1983 and 1995–1996), smallerthan 1%. The steady-state concentration profiles of particular metalsversus salinity are fairly constant in the coastal-estuarine and marinewatermasses, but decrease very abruptly from the first to the secondwatermass. Assuming a conservative behaviour of the particular metals,offshore fluxes and the resulting concentration increases agree fairly wellwith the observed values. The ratios of the Scheldt input to the particulatetrace metal offshore flux vary between 30 to 46% (1981–1983)and 13 to 37% (1995–1996). The contribution of the Scheldtestuary to the flows parallel to the coast ranges from 1.6 to 2.9%(1981–1983) and from 0.6 to 1.6% (1995). This revised version was published online in July 2006 with corrections to the Cover Date.     

GENETIC DIVERSITY WITHIN BALTIC SEA POPULATIONS OF NODULARIA (CYANOBACTERIA)1

The determination of the genetic structure of microbial populations has, until recently, required the establishment of many independent clonal cultures for genotypic analysis. In such studies it has been necessary to assume that isolates able to grow in laboratory culture are representative of the full range of diversity within the natural population. In order to test this assumption we used the polymerase chain reaction (PCR) to amplify the intergenic spacer region of the Phycocyanin operon (PC-IGS) from filaments of Nodularia taken both from clonal cultures and from natural populations in the Baltic Sea. Analysis of the nucleotide sequences revealed more variation among 16 cultured isolates than within 23 single filaments sampled from a natural population. As a means of rapidly determining population genetic structure we designed and used mixtures of allele-specific amplification primers in diagnostic PCRs to identify which PC-IGS allele was present in single filaments from natural cyanobacterial assemblages. Using this method, we determined the PC-IGS genotype of 156 filaments from 9 sampling stations throughout the central basin of the Baltic Sea in July 1996. Our results show that two distinct genotypes of Nodularia are present in the population at all stations. Although the two types were present in approximately equal numbers, they were not distributed uniformly.     

Electron microprobe analysis of juvenile walleye pollock,Theragra chalcogramma,otoliths from Alaska: a pilot stock separation study

Synopsis The incorporation of dissolved oceanic constituents in the otoliths of fish has potential as a chemical tracer for reconstructing the early life history of marine fish. Wavelength dispersive spectrometers on an electron microprobe were used to measure Na, Mg, P, S, Cl, K, Ca, and Sr concentrations on the outer margins of 57 juvenile walleye pollock, Theragra chalcogramma, otoliths from five locations in the Gulf of Alaska and Bering Sea. Discriminant analyses that used various combinations of Na, P, K, Sr, and fish standard length and/or age showed that 60–80% of the samples could be assigned to the correct capture locality. While the concentrations of some of the measured elements correlated with standard length or age of the fish, there are measurable differences among localities when concentrations are length or age corrected, mainly due to differences in Na and K concentrations. Elemental composition of otoliths potentially could be used to assign fish from a mixed stock fishery to original stocks, information that is greatly needed for the effective management of fish stocks.     

Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II

The conformation of the repeating heptapeptide unit of the carboxyl-terminal domain of RNA Polymerase II, Y¹S²P³T⁴S⁵P⁶S⁷ has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56-residue peptide H₂N-(S²P³T⁴S⁵P⁶S⁷Y^l)₈-COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β-turns located at Ser²-Pro³-Thr⁴-Ser⁵ (SPTS) and Ser⁵-Pro⁶-Ser⁷-Tyr¹ (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry-simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56-mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56-mer. Twenty-seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β-turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser², Ser⁵, Thr⁴, and Ser⁷ all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl-terminal domain with a regular but unusual secondary structure consisting of a series of staggered β-turns. © 1995 Wiley-Liss, Inc.     

Homology modeling by the ICM method

Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the noncon-served portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed. © 1995 Wiley-Liss, Inc.     

Crosslinking of uteroglobin by transglutaminase

Uteroglobin, a progesterone induced, pregnancy related protein, can be incorporated into higher molecular weight proteins by human placental Factor XIIIa. This process is time dependent, requires CaCl2 and can be inhibited by the addition of polylysine, dansylcadavarine or histamine. Crosslinking of uteroglobin into higher molecular weight proteins can also be brought about by guinea pig liver transglutaminase. Such a process may be involved in the modification of epididymal spermatozoa to suppress their antigenicity.     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.