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Lakes are generally supersaturated in carbon dioxide (CO2) and emitters of CO2to the atmosphere. However, estimates of CO2flux (FCO2) from lakes are seldom based on direct flux measurements and usually do not account for nighttime emissions, yielding risk of biased assessments. Here, we present direct FCO2measurements from automated floating chambers collected every 2–3 hr and spanning 115 24 hr periods in three boreal lakes during summer stratification and before and after autumn mixing in the most eutrophic lake of these. We observed 40%–67% higher mean FCO2in daytime during periods of surface water CO2supersaturation in all lakes. Day‐night differences in wind speed were correlated with the day‐night FCO2differences in the two larger lakes, but in the smallest and most wind‐sheltered lake peaks of FCO2coincided with low‐winds at night. During stratification in the eutrophic lake, CO2was near equilibrium and diel variability of FCO2insignificant, but after autumn mixing FCO2was high with distinct diel variability making this lake a net CO2source on an annual basis. We found that extrapolating daytime measurements to 24 hr periods overestimated FCO2by up to 30%, whereas extrapolating measurements from the stratified period to annual rates in the eutrophic lake underestimated FCO2by 86%. This shows the importance of accounting for diel and seasonal variability in lake CO2emission estimates. Considerable carbon cycling occurs within lakes, and carbon inputs from the catchment can be processed internally, stored in sediment and biomass or transported downstream. Additionally, carbon is exchanged with the atmosphere, resulting in lake uptake or atmospheric emission of carbon dioxide. Carbon dioxide exchanges from lakes have globally significant implications, but may be highly variable in time in ways that are not yet accounted for in emission estimates. Here, we estimated carbon dioxide exchange during multiple days and nights in three lakes with different nutrient levels during summer and autumn. For the most nutrient rich lake we also covered the period of water column mixing in autumn, which constitutes a critical time for carbon exchange. When carbon dioxide emission exceeded uptake, we found 40%–67% higher average exchange rates during daytime than nighttime. In contrast, the most nutrient rich lake experienced clear day‐night differences only after autumn mixing, when carbon dioxide emissions increased. We found that only using daytime measurements overestimated carbon dioxide emissions by up to 30%, and not accounting for autumn mixing underestimated carbon dioxide emissions by 86%. This shows the importance of accounting for day‐night and seasonal variability in lake carbon dioxide emission estimates. CO2emission was measured at 2–3 hr resolution for 1–2 months in three boreal lakes, using multiple automated floating chambersConsistently higher daytime emission was observed in all lakes, but in the eutrophic lake diel variability was limited to post‐lake mixingAccounting for diel variability of CO2emission within stratified and mixed periods is important to improve estimates of aquatic emissions CO2emission was measured at 2–3 hr resolution for 1–2 months in three boreal lakes, using multiple automated floating chambers Consistently higher daytime emission was observed in all lakes, but in the eutrophic lake diel variability was limited to post‐lake mixing Accounting for diel variability of CO2emission within stratified and mixed periods is important to improve estimates of aquatic emissions

C-reactive protein (CRP) is a major acute phase protein in humans and rabbits. Its synthesis by the liver varies over a 1000-fold range depending on the presence and severity of inflammatory stimuli. In previous studies of synthesis and secretion of rabbit CRP, we showed that secretion becomes more efficient over the course of the acute phase response as CRP synthesis rates increase (Macintyre, S.S., Kushner, I., and Samols, D. (1985) J. Biol. Chem. 260, 4169-4173). The current studies were undertaken to help distinguish between two alternative explanations for this finding: 1) that secretion efficiency may simply be a property of the rate of synthesis and intracellular concentration of CRP or 2) that secretion may be regulated by separate intracellular mechanisms. A fusion gene containing the mouse metallothionein I promoter linked to the protein coding region of the rabbit CRP gene was introduced into the human hepatoma cell line, NPLC, and the nonliver cell line, HeLa. In this system a graded response of the mouse metallothionein I promoter following exposure to increasing zinc concentrations results in increasing CRP synthesis. Unlike hepatocytes from rabbits undergoing the acute phase response, we found that rabbit CRP was secreted by these transfected cell lines with a very high degree of efficiency which was independent of the rate of CRP synthesis. This finding implies that normal rabbit hepatocytes retard the secretion of CRP and that this inhibition is diminished as the acute phase response progresses. It further indicates that the relationship between changes in synthetic rate and efficiency of secretion of rabbit CRP is not a causal one and that synthesis and secretion of CRP by rabbit hepatocytes are regulated by independent intracellular mechanisms during the acute phase response.

We studied the kinetics of synthesis and secretion of the acute phase plasma protein, C-reactive protein, in primary hepatocyte cultures prepared from rabbits manifesting differing degrees of the acute phase response to inflammatory stimulus. In cultures prepared from progressively more responsive animals, rate of C-reactive protein secretion increased to a much greater degree than did intracellular C-reactive protein content, resulting in a progressive decrease in the ratio of intracellular content to rate of secretion. This ratio, which represents the time required to secrete the amount of C-reactive protein contained within the intracellular pool, decreased from 18 h in cultures from unstimulated rabbits to 2.5 h in cells from highly responsive animals. In contrast, these ratios for albumin were short and fell within a narrow range (0.8-2.1 h). In pulse-chase labeling experiments, the time required for secretion of 50% of pulse-labeled C-reactive protein varied markedly, ranging from well over 6 h in cells from a minimally responsive animal to about 75 min in cells from a highly responsive rabbit. In contrast, the half-time for secretion of albumin was consistently about 45 min in the same cultures. Taken together, these findings indicate that the process by which C-reactive protein is secreted becomes more efficient during the course of the acute phase response. Recent studies have indicated that secretory proteins pass from the rough endoplasmic reticulum to Golgi at different and characteristic rates, possibly by a receptor-mediated process in which rate of transfer is determined by receptor affinity. We postulate that C-reactive protein secretion is regulated, during the course of the acute phase response, either by alterations in availability of specific receptors or by competition between different secretory proteins for a common receptor.

The distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species was examined. Enzyme activity was demonstrated in cell-free growth media from all members of the family Vibrionaceae which were tested except Plesiomonas shigelloides. In each case, enzyme was produced in exponential to early stationary phase and was excluded from Sepharose 6B, indicating a complex of high molecular weight. In a limited survey of other families, Stahylococcus aureus was the only organism outside the Vibrionaceae which was shown to produce the enzyme. In this case, however, the enzyme exhibited much less activity against erythrocyte membranes and appeared to have a lower molecular weight. The reasons for these differences and the importance of the acyltransferase as a biochemical identification tool are discussed.

The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids. A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively. In the first case, the lipophilic sequence anchored the protein in the plasma membrane. In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor. By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence. Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4. Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA. Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly.

The signal sequence of the precursor of the Escherichia coli outer membrane protein OmpA was altered by oligonucleotide insertions into the corresponding gene. In one case, OmpA-S1, the hydrophobic core of the signal peptide, is reduced from 12 to 10 residues, and one positive charge is added near the NH2-terminus. In another case, OmpA-P1, the hydrophobic core is extended from 12 to 16 residues. The pro-OmpA protein is normally processed partially co- and partially posttranslationally. Processing of the pro-OmpA-S1 protein was entirely posttranslational and that of the pro-OmpA-P1 protein strictly cotranslational. Evidence is presented which strongly suggests that posttranslational processing reflects posttranslational translocation across the plasma membrane. The generation times of cells expressing pro-OmpA-P1 or pro-OmpA-S1 were identical and the pro-OmpA-S1 polypeptide could be chased into the mature protein in the absence of protein synthesis. Hence, it does not matter which mode of processing, or rather translocation, is used. The same oligonucleotides were inserted into the ompA gene of plasmid pRD87; a plasmid which leads to overproduction of the protein and to massive accumulation of both the mature protein and the precursor. In the OmpA signal sequence encoded by pRD87-P2 the hydrophobic core is extended from 12 to 20 residues. This peptide was also rapidly removed. Therefore, regardless of whether the hydrophobic core contains 12, 16, or 20 lipophilic residues, not only does the signal sequence always function correctly to mediate export, but in each case, the cleavage site is always accessible to the signal peptidase.

C-reactive protein (CRP) mRNA was assayed by cell-free translation of poly(A)-containing liver RNA isolated both from rabbits stimulated to undergo the acute-phase response and from unstimulated control rabbits. No CRP-related translation products were identified until the denaturant methylmercury hydroxide (CH3HgOH) was added to the RNA before cell-free translation. In the presence of the denaturant, a 24000-Da translation product was synthesized which was immunochemically identifiable as the CRP primary translation product. It is likely that rabbit CRP mRNA can form a stable intramolecular duplex which interferes with its translatability in vitro. The 24000-Da CH3HgOH-facilitated cell-free translation product was not detected in poly(A)-containing liver RNA from unstimulated animals, indicating that the concentration of translatable CRP mRNA was dramatically induced during the acute-phase response. On the basis of absorption experiments, the 24000-Da CRP primary translation product was immunochemically more closely related to denatured CRP than to native CRP.

The synthesis and secretion of the acute-phase protein C-reactive protein by rabbit primary hepatocyte cultures was investigated. Hepatocytes prepared from animals that had received inflammatory stimuli 18-24 h before cell isolation were found to incorporate radiolabelled amino acids into C-reactive protein throughout the 48 h culture period. Intracellular C-reactive protein was found to be in steady state and there was no significant degradation of extracellular C-reactive protein, permitting direct estimation of rate of synthesis from rate of extracellular accumulation. Both C-reactive protein and total secreted protein were synthesized at constant rates for at least 24 h in culture. Mean rate of accumulation of newly synthesized total proteins in medium of cultures from six stimulated animals was 40% greater than was found in cultures from nine control (unstimulated) animals; this difference did not achieve statistical significance (0.05 less than P less than 0.10). Mean rate of C-reactive-protein synthesis represented 3.9% of total secreted-protein synthesis in cultures prepared from stimulated animals compared with 0.3% in cultures from control animals (P less than 0.001). Further, there was a correlation between C-reactive-protein synthesis by cultured hepatocytes and serum C-reactive-protein concentration at time of hepatocyte isolation (P less than 0.001). Rates of C-reactive-protein synthesis by hepatocyte cultures from stimulated animals were in good agreement with those previously measured in isolated perfused livers and those calculated from results of studies in vivo.

The Central Clydeside Conurbation (CCC) has relatively high mortality rates. This paper examines whether it also has relatively high rates of ill health, using data from three cohorts (aged 15, 35 and 55 in 1987/88) in the West of Scotland. Comparisons on a range of self-reported physical and mental health indicators, anthropometric measures, blood pressure, and respiratory function were made with comparable age groups in ten British or Scottish national studies. The older two cohorts in the CCC exhibited relatively high rates of longstanding and limiting longstanding illness and the youngest cohort had relatively poor psychosocial health, compared to their age peers elsewhere. Fewer differences were found in blood pressure, anthropometric measures or respiratory function although older CCC residents were slightly shorter than in Britain as a whole and had slightly poorer respiratory function. Central Clydesiders in the late 1980s were generally in poorer health than those of the same sex and similar age elsewhere in the UK, but the extent of the disadvantage varied across different dimensions of health, and was not as marked as some stereotypes of the West of Scotland would suggest.

The importance of the two major extracellular enzymes of Aeromonas salmonicida, glycerophospholipid: cholesterol acyltransferase (GCAT) and a serine protease (AspA), to the pathology and mortality of salmonid fish with furunculosis had been indicated in toxicity studies. In this study, the genes encoding GCAT (satA) and AspA (aspA) have been cloned and mutagenized by marker replacement of internal deletions, and the constructs have been used for the creation of isogenic satA and aspA mutants of A. salmonicida. A pSUP202 derivative (pSUP202sac) carrying the sacRB genes was constructed to facilitate the selection of mutants. The requirement of serine protease for processing of pro-GCAT was demonstrated. Processing involved the removal of a short internal fragment. Surprisingly, pathogenicity trials revealed no major decrease in virulence of the A. salmonicida delta satA::kan or A. salmonicida delta aspA::kan mutants compared to the wild-type parent strains when Atlantic salmon (Salmo salar L.) were challenged by intraperitoneal injection. Moreover, using a cohabitation model, which more closely mimics the natural disease, there was also no significant decrease in the relative cumulative mortality following infection with either of the deletion mutants compared to the parent strain. Thus, although these two toxins may confer some competitive advantage to A. salmonicida, neither toxin is essential for the very high virulence of A. salmonicida in Atlantic salmon. This first report of defined deletion mutations within any proposed extracellular virulence factor of A. salmonicida raises crucial questions about the pathogenesis of this important fish pathogen.

The distal part of the long tail fibers of the Escherichia coli phage T4 consists of a dimer of protein 37. A fragment of the corresponding gene, encoding 253 amino acids, was inserted into several different sites within the cloned gene for the 325-residue outer membrane protein OmpA. In plasmid pTU T4-5 the fragment was inserted once and in pTU T4-10 tandemly twice between the codons for residues 153 and 154 of the OmpA protein. In pTU T4-22 two fragments were present, in tandem, between the codons for residues 45 and 46 of this protein. In pIN T4-6 one fragment was inserted into the ompA gene immediately following the part encoding the signal sequence. The corresponding mature proteins consist, in this order, of 605, 860, 835, and 279 amino acid residues. All precursor proteins were processed and translocated across the plasma membrane. Hence, not only can the OmpA protein serve as a vehicle for export of a nonsecretory protein, but the signal sequence alone can also mediate export of such a protein. Export of the pro-OmpA protein depends on the SecA protein. Export of the tail fiber fragment expressed from pIN T4-6 remained SecA dependent. Thus, the secA pathway in this case is chosen by the signal peptide. It is proposed that a signal peptide can mediate translocation of nonsecretory proteins as long as they are export-compatible. The inability of a signal sequence to mediate export of some proteins appears to be due to export incompatibility of the protein rather than to the absence of information, within the mature part of the polypeptide, which would be required for translocation.

The Pseudomonas aeruginosa polyvalent vaccine PEV and its 16 constituent monovalent extracts from International Antigenic Typing System serotypes 1 through 13 and 15 through 17 (J. J. Miler, J. F. Spilsbury, R. J. Jones, E. A. Roe, and E. J. L. Lowbury, J. Med. Microbiol. 10:19-27, 1977) were subjected to biochemical analysis and to detailed immunochemical analysis with rabbit anti-PEV immunoglobulins. The results of chemical analysis, of analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed in conjunction with silver staining, and of analysis by crossed immunoelectrophoresis, sodium dodecyl sulfate-polyacrylamide gel-crossed immunoelectrophoresis, and Western blotting showed clearly that lipopolysaccharide was a major constituent of each monovalent extract and that it was probably the dominant antigen present in at least 15 of the 16 monovalent extracts. A 16.2-kilodalton protein, which was pronase resistant and nonsedimentable at 105,000 X g and which appeared to be biochemically and antigenically unrelated to pili, was a common although minor antigen for all extracts. Several other proteins, some of outer membrane origin, were also detected in unformalinized extracts, but these were also minor antigenic constituents of the vaccine. Neither pilin nor flagellin appeared to be major protein constituents of tested monovalent extracts, although anti-flagella antibodies could be demonstrated in rabbit anti-PEV by Western blotting. Preliminary analysis by crossed immunoelectrophoresis of serum raised in volunteers to PEV also indicated the presence therein of antibodies to lipopolysaccharide antigens.

The nature of the protective antigen in one of the sixteen monovalent extracts (viz., extract-6) contributing to the pseudomonas polyvalent extract vaccine (PEV) was studied in a mouse challenge assay. Selective removal, by filtration through Sep-Pak C18 cartridges, of two major protein antigens with molecular weights of 16,200 and 21,000 had no effect on the protection afforded by extract-6. When analyzed on the basis of 2-keto-3-deoxyoctonate, lipopolysaccharide (LPS) purified by hot phenol extraction (LPS-A) from Pseudomonas aeruginosa (International Antigenic Typing System serotype 6) could account in full for the protective capacity of extract-6. Comparative analysis of LPS heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that both extract-6 and LPS-A possessed similar spectra of smooth LPS molecules, containing between 10 and approximately equal to 50 O-antigen repeating units. Differences in the profiles of heterogeneity displayed by LPS in LPS-A and extract-6 were restricted to molecular species with short O-antigen chains. Subfractionation of LPS molecules on the basis of number of O-antigen repeating units was achieved by gel filtration in the presence of deoxycholate. Protection experiments performed on the subfractionated species of LPS-A revealed a relationship between O-antigen chain length and protective capacity; molecules with over 18 O-antigen repeating units being 50 to 100 times more protective than those with zero-two repeating units. The results indicate that most of the protection afforded by LPS-A and extract-6 can be accounted for by LPS molecules possessing extended (10 or more) O-antigen repeating units.

The membrane part of the 325-residue outer membrane protein OmpA of Escherichia coli encompasses residues 1-177. This part is thought to cross the membrane eight times in antiparallel beta-strands, forming four loops of an amphipathic beta-barrel. With the aim of gaining some insight into the mechanism of sorting, i.e. the way the protein recognizes and assembles into its membrane, a set of point mutants in the ompA gene has been generated. Selection for toxicity of ompA expression following mutagenesis with sodium bisulfite yielded genes with multiple base pair substitutions, the majority of which resulted in amino acid substitutions in the membrane moiety of the protein. None of the altered proteins was blocked in membrane incorporation. A proline residue exists at or near each of the presumed turns at the inner side of the outer membrane. Using oligonucleotide-directed mutagenesis, each of them was replaced by a leucine residue which is thought to be a turn blocking residue. None of these proteins had lost the ability to be incorporated into the membrane. Apparently, leucine residues are tolerated at turns in this protein. To interfere with the formation of antiparallel beta-strands, four double mutants were prepared: ompA-ON3 (Ala11—-Pro, Leu13—-Pro), -ON4 (Ala11—-Asp, Leu13—-Pro), -ON5 (Gly160—-Val, Leu162—-Arg), and -ON6 (Leu164—-Pro, Val166—-Asp). The former three proteins and even quadruple mutants consisting of a combination of ompA-ON2 or -ON4 with -ON5 were not defective in membrane assembly. In contrast, the OmpA-ON6 protein was translocated across the plasma membrane but could not be incorporated into the outer membrane. It is concluded that at least one rather small area of the polypeptide is of crucial importance for the assembly of OmpA into the outer membrane.

A series of overlapping deletions has been constructed in the ompA gene which encodes the 325-residue Escherichia coli outer membrane protein OmpA. Immunoelectron microscopy showed that the OmpA fragments were either located in the periplasmic space or were associated with the outer membrane. Apparently an area between residues 154 and 180 is required for this association; all proteins missing this area were found to be periplasmic. The nature of this association remained unknown; no membrane-protected tryptic fragments could be identified for any of these polypeptides. Hybrid genes were constructed encoding parts of the periplasmic maltose binding protein and an area of the ompA gene coding for residues 154-274. The corresponding proteins were not localized to the outer membrane but remained attached to the outer face of the plasma membrane, possibly because the normal mechanism of release from this membrane was impaired. In the OmpA protein the conspicuous sequence Ala180-Pro-Ala-Pro-Ala-Pro-Ala-Pro187 exists. Frameshift mutants were constructed to eliminate this sequence. There was no effect on the incorporation of the mutant proteins into the outer membrane. Thus, this “hinge” region is not involved in sorting. A proposal suggesting the existence of a sorting signal common to several outer membrane proteins (Benson, S. A., Bremer, E., and Silhavy, T. J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3830-3834) was subsequently rejected (Bosch, D., Leunissen, J., Verbakel, J., de Jong, M., van Erp, H., and Tommassen, J. (1986) J. Mol. Biol. 189, 449-455; Freudl, R., Schwarz, H., Klose, M., Movva, N. R., and Henning, U. (1985) EMBO J. 4, 3593-3598). Although it is not known whether or not the outer membrane association observed represents a step in the normal sorting mechanism, it is concluded that it remains an open question whether or not a sorting signal, as proposed originally, exists in outer membrane proteins.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.

Human erythrocyte membrane glycerophospholipids are deacylated by Aeromonas hydrophila 13-h culture supernatants, resulting in the production of cholesterol ester, free fatty acid, and water-soluble phosphates. This activity appears to be due to the actions of an acyltransferase (phosphatide:cholesterol acyltransferase, EC 2.3.1 group) and a phospholipase (phosphatide acyl-hydrolase). The enzyme activities are produced simultaneously in late exponential/early stationary phase, are precipitated together from the culture supernatant with 85% ammonium sulfate, and are eluted together near the void volume during gel filtration on Sepharose 6B. These results suggest that A. hydrophila produces a multienzyme complex with an unusual mode of action on membrane lipids. The complex is distinct from the hemolytic factor aerolysin, which is also produced by A. hydrophila.

A glycerophospholipid:cholesterol acyltransferase has been purified to near homogeneity from cell-free culture supernatants of Aeromonas salmonicida. The characteristics of the enzyme distinguish it from bacterial phospholipases; however, it shares several properties with the lecithin:cholesterol acyltransferase of mammalian plasma. Thus, the enzyme inhibits 2-positional specificity as an acyltransferase and it will act as a phospholipase A2 in the absence of cholesterol. Furthermore, it has no divalent cation requirement and it is stimulated both by albumin and by human apolipoprotein A-I. Unlike the mammalian acyltransferase, however, the bacterial enzyme is not specific for phosphatidylcholine and in addition it can use human erythrocyte membranes as substrates. Similar to Naja naja phospholipase A2, it acts asymmetrically on intact erythrocytes.

This report, the fourth of a related series dealing with the isotopic analysis of uranium, describes the use of a hollow cathode source and a 4-m, direct-reading, Bausch and Lomb high-resolution spectrograph. A relative precision of ±3.0%, expressed at the 95% confidence level, was obtained with naturally occurring uranium for a single cathode determination of U235measured in twenty min. Evidence for a weak uranium line structure interfering with the U235measurement is presentedMinor isotopes, U231and U236, present up to a few percent at the higher U235concentrations, did not interfere with U235measurements. Measurements of U234, however, could not be made in the presence of high U235concentrations, thereby limiting the determination of total uranium necessary for calculating the percent U235. At the lower U235concentrations, the concentrations of minor isotopes are generally too small to significantly affect the total uranium content of the sample.

Evidence shows that where we live has a significant impact on our mental health. [ABSTRACT FROM AUTHOR]