The characteristics of macrophage-like cell lines derived from normal sheep spleens

Macrophage-like cell lines were derived from sheep spleens using conditioned medium from L-929 mouse cells as a source of colony stimulating factor. In seven out of ten attempts colonies of macrophage-like cells appeared after 2-3 weeks of culture. The cells were established in culture as cell lines, and survived 120 passages. They were strongly (+ +) positive for non-specific esterase but negative for peroxidase and produced detectable but small amounts of lysozyme (0.21-1.76 micrograms/10(6) cells). Latex particles were actively phagocytosed. Bacteria (Staphylococcus albus, Staphylococcus aureus) attached to the cell surface and were internalized in the presence of specific antibody. Expression of receptors for immunoglobulin and complement varied somewhat between the different cell lines: the proportion of receptor-bearing cells ranged between 9 and 26% FC-receptors, and 10 and 38% for C-receptors. The cell lines displayed a peculiar karyotype as well as protein profile that were different from normal sheep but similar between the different cell lines. Culture supernatants of the cell lines contained a colony stimulating activity which was used to establish further cell lines. They also spontaneously produced an interleukin-1-like activity that had no effect on baseline proliferation of sheep lymphocytes but enhanced their response to PHA (1.7-fold) particularly in conjunction with sheep IL-2 (4-fold). Prostaglandin E2 was produced in a growth-cycle dependent manner: the peak production occurred on the second day (77-140 pg/ml) at 2 x 10(5) cells and declined to 33-50 pg/ml on the eighth day when cell numbers had increased to 2-3 x 10(6). These easily cultured cell lines derived from normal tissue without the introduction of viral DNA should provide a useful source of material for studies of macrophage function in sheep.     

Primary structure of cold-adapted alkaline phosphatase from a Vibrio sp. as deduced from the nucleotide gene sequence

Alkaline phosphatases (AP) are widely distributed in nature, and generally have a dimeric structure. However, there are indications that either monomeric or multimeric bacterial forms may exist. This paper describes the gene sequence of a psychrophilic marine Vibrio AP, previously shown to be particularly heat labile. The kinetic properties were also indicative of cold adaptation. The amino acid sequence of the Vibrio G15-21 AP reveals that the residues involved in the catalytic mechanism, including those ligating the metal ions, have precedence in other characterized APs. Compared with Escherichia coli AP, the two zinc binding sites are identical, whereas the metal binding site, normally occupied by magnesium, is not. Asp-153 and Lys-328 of E. coli AP are His-153 and Trp-328 in Vibrio AP. Two additional stretches of amino acids not present in E. coli AP are found inserted close to the active site of the Vibrio AP. The smaller insert could be accommodated within a dimeric structure, assuming a tertiary structure similar to E. coli AP. In contrast the longer insert would most likely protrude into the interface area, thus preventing dimer formation. This is the first primary structure of a putative monomeric AP, with indications as to the basis for a monomeric existence. Proximity of the large insert loop to the active site may indicate a surrogate role for the second monomer, and may also shape the catalytic as well as stability characteristics of this enzyme.     

Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.     

The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.     

Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli

Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal-binding sites in the active site of APs, and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs. This may explain their different catalytic efficiencies, as the Mg2+ most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116/Lys274, and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116/Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge. Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold-active Vibrio AP both locally and globally, and this influences kinetic properties.     

Microscopic rate-constants for substrate binding and acylation in cold-adaptation of trypsin I from Atlantic cod

Temperature imposes limits on where life can thrive and this is evident in the evolution of the basic structural properties of proteins. Cold-adaptation of enzymes is one example, where the catalytic rate constant (k(cat)) is increased compared with hot-acclimated homologous under identical assay conditions. Trypsin I from Atlantic cod (Gadus morhua) has catalytic efficiency (k(cat)/K(m)) for amide hydrolysis that is 17-fold larger than observed for bovine trypsin. Here, the individual rate-constants for association of substrate (k(1)), dissociation of substrate (k(-1)), and acylation of the enzyme (k(2)) have been determined using benzoyl-Arg-p-nitroanilide or benzyloxycarbonyl-Gly-Pro-Arg-p-nitroanilide as substrates. Rather unexpectedly, by far the largest difference (37-fold increase) was observed in k(1), the rate constant for binding of substrate. The cold-adaptation of the dissociation and catalytic steps were not as prominent (increased by 3.7-fold). The length of substrate did have an effect by increasing the reaction rate by 70-fold, and again, the step most affected was the initial binding-step.     

γ-l-Glutamyl-l-pipecolic acid in Gleditsia caspica

y-l-Glutamyl-l-pipecolic acid has been isolated from seeds of Gleditsia caspica (L.) Desf. Proof of its structure was obtained by chromatographic and spectroscopic examination of the natural product and its hydrolytic products. The new compound is the first example of a naturally occurring γ-glutamyl imino acid.     

Severity of Influenza A 2009 (H1N1) Pneumonia Is Underestimated by Routine Prediction Rules. Results from a Prospective,Population-Based Study

Background

Characteristics of patients with community-acquired pneumonia (CAP) due to pandemic influenza A 2009 (H1N1) have been inadequately compared to CAP caused by other respiratory pathogens. The performance of prediction rules for CAP during an epidemic with a new infectious agent are unknown.

Methods

Prospective, population-based study from November 2008–November 2009, in centers representing 70% of hospital beds in Iceland. Patients admitted with CAP underwent evaluation and etiologic testing, including polymerase chain reaction (PCR) for influenza. Data on influenza-like illness in the community and overall hospital admissions were collected. Clinical and laboratory data, including pneumonia severity index (PSI) and CURB-65 of patients with CAP due to H1N1 were compared to those caused by other agents.

Results

Of 338 consecutive and eligible patients 313 (93%) were enrolled. During the pandemic peak, influenza A 2009 (H1N1) patients constituted 38% of admissions due to CAP. These patients were younger, more dyspnoeic and more frequently reported hemoptysis. They had significantly lower severity scores than other patients with CAP (1.23 vs. 1.61, P = .02 for CURB-65, 2.05 vs. 2.87 for PSI, P<.001) and were more likely to require intensive care admission (41% vs. 5%, P<.001) and receive mechanical ventilation (14% vs. 2%, P = .01). Bacterial co-infection was detected in 23% of influenza A 2009 (H1N1) patients with CAP.

Conclusions

Clinical characteristics of CAP caused by influenza A 2009 (H1N1) differ markedly from CAP caused by other etiologic agents. Commonly used CAP prediction rules often failed to predict admissions to intensive care or need for assisted ventilation in CAP caused by the influenza A 2009 (H1N1) virus, underscoring the importance of clinical acumen under these circumstances.     

Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea

Alkaline phosphatase (AP) from Atlantic cod (Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6-8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity.     

Amino acid sequence of the cold-active alkaline phosphatase from Atlantic cod (Gadus morhua)

Atlantic cod is a marine fish that lives at low temperatures of 0-10 degrees C and contains a cold-adapted alkaline phosphatase (AP). Preparations of AP from either the lower part of the intestines or the pyloric caeca area were subjected to proteolytic digestion, mass spectrometry and amino acid sequencing by Edman degradation. The primary structure exhibits greatest similarity to human tissue non-specific AP (80%), and approximately 30% similarity to AP from Escherichia coli. The key residues required for catalysis are conserved in the cod AP, except for the third metal binding site, where cod AP has the same variable residues as mammalian APs (His153 and His328 by E. coli AP numbering). General comparison of the amino acid composition with mammalian APs showed that cod AP contains fewer Cys, Leu, Met and Ser, but proportionally more Asn, Asp, Ile, Lys, Trp and Tyr residues. Three N-linked glycosylation sites were found. The glycan structure was determined as complex biantennary in type with fucose and sialic acid attached, although a trace of complex tri-antennary structure was also observed. A three-dimensional model was obtained by homology modelling using the human placental AP scaffold. Cod AP has fewer charged and hydrophobic residues, but more polar residues at the intersubunit surface. The N-terminal helix arm that embraces the second subunit in dimeric APs may be more flexible due to a replaced Pro at its base. One disulfide bridge was found instead of the two present in most other APs. This may invoke greater movement in the structure that together with weaker subunit contacts leads to improved catalytic efficiency.