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.

Properties of elastase from Atlantic cod, a cold-adapted proteinase.

An intestinal elastase was purified from Atlantic cod (Gadus morhua) of apparent molecular mass 24.8 kDa as determined by SDS-PAGE and with isoelectric point above pI 9.3. Heat stability and stability towards acidic pH was reduced in the cod enzyme as compared with porcine intestinal elastase. N-terminal amino-acid sequence analysis of cod elastase showed considerable similarity with porcine elastase. The cod enzyme was less sensitive to phenylmethylsulfonyl fluoride inhibition than porcine elastase, but sensitivity towards other inhibitors was similar. Kinetic properties were examined using the substrate Suc-Ala-Ala-Ala-p-nitroanilide and the cod enzyme was found to have more than 2-times turnover rate (kcat) as compared with the porcine enzyme, and slightly higher Km values. Thus, the catalytic efficiency (kcat/Km) of Atlantic cod elastase was about 2-times higher than observed with porcine elastase, which indicates an adaptive response towards the low temperature environmental in which the cod lives. Substrate specificity was studied by digestion of oxidized B-chain of insulin and by using synthetic substrates. Digestion was most rapid at the carbonyl side of alanine residues, but also occurred at valine and leucine residues.

Structure of chymotrypsin variant B from Atlantic cod, Gadus morhua.

The amino-acid sequence of chymotrypsin variant B isolated from the pyloric caeca of Atlantic cod has been elucidated. The characterization of the primary structure is based on N-terminal Edman degradation and mass spectrometry of the native protein and enzymatically derived peptides. Chymotrypsin variant B showed 72% sequence identity with the A-variant and 64% and 62%, respectively, with the bovine counterparts A and B, all consisting of 245 amino acids. This new sequence contains a higher proportion of charged residues compared with bovine chymotrypsin but fewer polar hydrogen-bond forming residues which might contribute to its lower thermostability. It also shares the emerging characteristics of other fish serine proteinases which have relatively higher methionine content, including a conserved Met-134 in a loop leading into a domain-connecting strand. The inherent mobility in methionine side-chains may contribute to the maintenance of flexibility at low temperatures. Several amino-acid sequence differences adjacent to the catalytic site are observed in the two cod chymotrypsin variants which also differ in kinetic properties. Unlike the mammalian chymotrypsins, which contain several autolysis sites, cod variant B only contains a single autolysis site. The three-dimensional structures of the A- and B-variants of cod has been modelled on the known crystal structure of bovine alpha-chymotrypsin showing almost superimposable structures.

Effects of different colloid infusions on ROTEM and Multiplate during elective brain tumour neurosurgery.

BACKGROUND: The European Medicines Agency does not recommend the use of hydroxyethyl starch-based volume replacement solutions in critically ill patients due to an increased risk of renal failure. However, this recommendation is questionable for its perioperative use. Several recent randomised controlled studies do not indicate a risk for renal failure-not even after high-risk surgery. Human albumin is used in our neurointensive care unit as a part of the "Lund concept" of brain injury resuscitation, and albumin has been introduced in elective neurosurgery instead of starch. The aim of our prospective unblinded observational cohort study was to compare the degree of dilutive coagulopathy after albumin and starch intra-operative fluid therapy. METHODS: Thirty-nine patients undergoing elective brain tumour surgery with craniotomy received either 130/0.42 hydroxyethyl starch or 5 % albumin infusions. The first 18 patients received starch, whereas the rest received albumin. Rotational thromboelastometry with ROTEM and platelet aggregometry with Multiplate were performed before surgery, after the first and second consecutive colloid infusions (250/500 ml albumin or 500/1000 ml starch) and at the end of surgery. RESULTS: Both intra- and inter-group comparisons showed more deranged ROTEM parameters after the higher doses of starch. Multiplate detected changes only in the albumin group after 500-ml infusion. Blood los did not differ between groups, nor did haemoglobin preoperatively or at end of surgery. Lower volumes of albumin were required to maintain stable intra-operative haemodynamic parameters; 250/500 ml albumin corresponded to 500/1000 ml starch. CONCLUSIONS: Hydroxyethyl starch affected coagulation at lower volumes, with a more prominent effect on clot structure at the end of surgery, corroborating previous research. Only albumin decreased platelet aggregation, and 5 % albumin had a more potential volume effect than 130/0.42 hydroxyethyl starch.

Calcium-dependent protein phosphorylation in bovine anterior pituitary membranes and intact cells.

A calmodulin-activated protein kinase has been identified in bovine anterior pituitary membranes. This enzyme phosphorylated one endogenous substrate of subunit molecular weight 53,000 in the membranes. Phosphorylation of this protein was rapid, was half-maximal at 2.5 microM calcium in the presence of saturating concentrations of calmodulin (CaM), and was inhibited by trifluoperazine and thioridazine. A second protein was phosphorylated by an endogenous protein kinase in anterior pituitary membranes. Phosphorylation of this 42,000 Mr protein was reduced by calcium, was independent of exogenously added CaM, and was increased by trifluoperazine or thioridazine. The 42,000 Mr protein may be the alpha-subunit of pyruvate dehydrogenase. Calcium-dependent protein phosphorylation was also observed in intact cells; the largest increases were seen in proteins of Mr 42,000, 21,000 and 17,000.

Effects of arterial and venous pressure alterations on transcapillary fluid exchange during raised tissue pressure.

OBJECTIVE: To assess local haemodynamic effects of raised tissue pressure per se and of arterial and venous pressure variations during raised tissue pressure, and to evaluate the vascular waterfall phenomenon. DESIGN: An isolated pump-perfused sympathectomised cat skeletal muscle enclosed in a plethysmograph. INTERVENTIONS: Hydrostatic capillary pressure (Pc), tissue volume alterations and blood flow were recorded at various arterial (PA) or venous (PV) pressure levels at a raised tissue pressure (Ptissue). Total vascular resistance (Rtot) and its three consecutive sections, arterial resistance (Rart), venular resistance (Rvenule), and venous outflow orifice resistance (Rorifice), were recorded. RESULTS: Increase in Ptissue increased Pc due to a marked increase in Rorifice and unchanged Rart and, when Ptissue > PV, by about 90% of the Ptissue increase. During the raised Ptissue, a PA increase (95 to 115 mmHg) increased Pc (by 3.1 +/- 1.1 mmHg) causing fluid filtration, and a PA decrease (95 to 75 mmHg) decreased Pc (by 2.4 +/- 0.5 mmHg) causing fluid absorption. Rart was unchanged, indicating impaired autoregulation. Increased PV had no haemodynamic effects when PV < Ptissue due to a gradual decrease in Rorifice towards zero when PV reached Ptissue. At PV > Ptissue blood flow and Pc increased gradually, causing fluid filtration. CONCLUSIONS: Tissue volume is increased by raised and decreased by lowered PA, the latter may be of use to decrease ICP in the injured brain. The results indicate that PEEP or head elevation will not influence ICP from the venous side if CVP < ICP. Finally, the "vascular waterfall phenomenon" was rejected as Rorifice is a normal variable fluid resistance.

A new therapy of post-trauma brain oedema based on haemodynamic principles for brain volume regulation.

OBJECTIVE: To evaluate a new therapy of posttraumatic brain oedema, with the main concept that opening of the blood-brain barrier upsets the normal brain volume regulation, inducing oedema formation. This means that transcapillary fluid fluxes will be controlled by hydrostatic capillary and colloid osmotic pressures, rather than by crystalloid osmotic pressure. If so, brain oedema therapy should include reduction of hydrostatic capillary pressure and preservation of normal colloid osmotic pressure. PATIENTS: 11 severely head injured comatose patients with brain swelling, raised intracranial pressure (ICP), and impaired cerebrovascular response to hyperventilation. INTERVENTIONS: To reduce capillary hydrostatic pressure the patients were given hypotensive therapy (beta 1-antagonist, metoprolol and alpha 2-agonist, clonidine) and a potential precapillary vasoconstrictor (dihydroergotamine). The latter may also decrease cerebral blood volume through venous capacitance constriction. Colloid osmotic pressure was maintained by albumin infusions. The concept implies the need of a negative fluid balance with preserved normovolaemia. RESULTS: ICP decreased significantly within a few hours of treatment with unaltered perfusion pressure in spite of lowered blood pressure. Of 11 patients 9 survived with good recovery/moderate disability, 2 died. This was compared to outcome in a historical control group with identical entry criteria, given conventional brain oedema therapy, where mortality/vegetativity/severe disability was 100%. CONCLUSION: The results indicate that the therapy should focus on extracellular rather than intracellular oedema and that ischemia is not the main triggering mechanism behind oedema formation. We suggest that our therapy is superior to conventional therapy by preventing herniation during the healing period of the blood-brain barrier.

On the role of monocytes/macrophages in the pathogenesis of central nervous system lesions in hereditary cystatin C amyloid angiopathy.

The pathogenesis of the deposition of a variant cystatin C as amyloid in hereditary cystatin C amyloid angiopathy (HCCAA) is not known. To address this question the synthesis and secretion of cystatin C in cultured monocytes from 9 carriers of the mutated cystatin C gene (5 symptomatic and 4 asymptomatic) was examined. The quantity of cystatin C in cells and supernatants was determined by the ELISA method, Western blots were done and selected samples immunostained for cystatin C. Monocytes from individuals carrying the gene defect synthesized cystatin C that was apparently not truncated, a form found in the cerebral amyloid deposits in HCCAA, but showed a distinctly lower rate of cystatin C synthesis than monocytes from healthy controls. The main difference was that the quantity of cystatin C was significantly lower in the supernatants in monocyte cultures from carriers of the gene defect than from healthy controls, possibly due to a partial block in its secretion. This abnormal processing of the cystatin C could explain the low cerebrospinal fluid levels of cystatin C in HCCAA and might be a part of the pathogenetic pathway of amyloid deposition. Furthermore it could, through a lower extracellular concentration of this inhibitor of cysteine proteinases, contribute to destruction of the amyloidotic blood vessels, leading to the most serious clinical manifestation in HCCAA, intracerebral hemorrhage.

Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.

Local vascular responses to elevation of an organ above the heart.

Elevation of an organ above the heart reduces the arterial and venous hydrostatic pressures in proportion to the height of elevation. Intact autoregulation protects organs, such as the brain and skeletal muscle, from significant alterations in blood flow and hydrostatic capillary pressure due to the decrease in arterial inflow pressure during such a manoeuvre. However, the consequences of the decreased hydrostatic pressure on the venous side are far from clarified. The present study analyses the local haemodynamic effects of the decrease in arterial and venous hydrostatic pressures that occur during vertical elevation of an organ above the heart at atmospheric and raised tissue pressures (0, 10 and 30 mmHg). A sympathectomized cat skeletal muscle enclosed in a plethysmograph and perfused from the animal was used as the experimental model. The results show that elevation of the muscle above the heart at atmospheric tissue pressure created a variable vascular resistance starting at the venous outlet of the organ, and related to the difference between tissue pressure and venous outflow pressure. This resistance completely protects the organ from the hydrostatic pressure alterations on the venous side. The results also show that arterial pressure variations will exert the same haemodynamic influences on the organ as tissue pressure variations, except for the formation of the venous outflow resistance at raised tissue pressure. The application of these results to normal and injured organs, e.g. normal and injured skeletal muscle and brain, with various tissue pressures, is discussed.

Propofol-fentanyl anesthesia compared to thiopental-halothane with special reference to recovery and vomiting after pediatric strabismus surgery.

Forty-four children, ASA physical status I or II, aged 1.5-14 years and admitted for strabismus surgery, were studied. The study compared the postoperative condition after two different anesthesia methods. All children were premedicated with midazolam rectally, received glycopyrrolate i.v. and were then randomised to one of two anesthetic methods: 1) induction with thiopental, maintenance with halothane or 2) induction with propofol supplemented with fentanyl, maintenance with propofol infusion. In both groups, tracheal intubation was performed after vecuronium i.v. and the children were ventilated manually. Peroperatively, patients receiving propofol/fentanyl had more episodes of bradycardia (P less than 0.001). Times to spontaneous breathing and extubation were shorter in the propofol/fentanyl group (P less than 0.05) and there was also a lesser degree of sedation during the first 2 h postoperatively (P less than 0.01). Fewer children in the propofol/fentanyl group vomited postoperatively (P less than 0.05). The apprehension score was higher in the propofol/fentanyl group compared to the thiopental/halothane group (P less than 0.05). We conclude that children undergoing strabismus surgery anesthetized with propofol/fentanyl had more episodes of peroperative bradycardia, a lower incidence of postoperative vomiting and a shorter recovery time, and were more apprehensive during the initial postoperative period than children anesthetized with thiopental/halothane.

Purification and characterization of trypsin from the poikilotherm Gadus morhua.

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.

Physiologic principles for volume regulation of a tissue enclosed in a rigid shell with application to the injured brain.

BACKGROUND: Preservation of a high cerebral perfusion (mean arterial) pressure to prevent ischemia has become the primary focus during treatment of severe head trauma because ischemia is favored as a triggering mechanism behind intracellular brain edema development and poor outcome. A high cerebral perfusion pressure, however, simultaneously may increase the hydrostatic vasogenic edema. The present paper evaluates the mechanisms behind the vasogenic edema by analyzing the physiologic hemodynamic mechanisms controlling the volume of a tissue that is enclosed in a rigid shell, possesses capillaries permeable for solutes, and has depressed autoregulation. RESULTS AND CONCLUSIONS: We contend that in the long run, the interstitial volume in such a tissue can be reduced only through reduction in arterial inflow pressure providing an otherwise optimal therapy to improve microcirculation. Therefore we argue, in contrast to the conventional view, that antihypertensive and antistress therapy may be of value by reducing the interstitial tissue volume during treatment of brain edema, and that the problem with ischemia during such therapy can be handled when considering an otherwise optimal intensive care. These physiologic principles of interstitial tissue volume regulation form the basic concept for the "Lund therapy" of severe head injuries, which is a new and controversial therapy of posttraumatic brain edema.

Aspects on the cerebral perfusion pressure during therapy of a traumatic head injury.

An actively raised cerebral perfusion pressure by vasopressors is nowadays often advocated during therapy of a post traumatic brain oedema to improve oxygenation of the brain. In this paper we argue that the arterial pressure not uncritically can be raised as the subsequent increase in hydrostatic capillary pressure may favour transcapillary filtration if the blood-brain barrier is opened for solutes. Further, the use of vasoconstrictor drugs to increase the perfusion pressure may in fact impair oxygenation to the penumbra zones around brain contusions but also to other tissues of the body, like the intestinal mucosa and the kidney. An alternative therapeutical concept which both ensures an adequate oxygenation of the brain and controls the intracranial pressure (ICP) is given. In short, it implies active antistress and sedative treatment, adequate fluid therapy with blood and colloids to normal haemoglobine and albumin values, artificial ventilation to normal PaCO2 and PaO2, and this in combination with antihypertensive and catecholamine reducing treatment with alpha 2-agonist and beta 1-antagonist.

Volume-targeted therapy of increased intracranial pressure: the Lund concept unifies surgical and non-surgical treatments.

Opinions differ widely on the various treatment protocols for sustained increase in intracranial pressure (ICP). This review focuses on the physiological volume regulation of the intracranial compartments. Based on these mechanisms we describe a protocol called 'volume-targeted' ('Lund concept') for treatment of increased ICP. The driving force for transcapillary fluid exchange is determined by the balance between effective transcapillary hydrostatic and osmotic pressures. Fluid exchange across the intact blood-brain barrier (BBB) is counteracted by the low permeability to crystalloids (mainly Na+ and Cl-) combined with the high osmotic pressure (5500 mmHg) on both sides of the BBB. This contrasts to most other capillary regions where the osmotic pressure is mainly derived from the plasma proteins (approximately 25 mmHg). Accordingly, the level of the cerebral perfusion pressure (CPP) is of less importance under physiological conditions. In addition cerebral intracapillary hydrostatic pressure (and cerebral blood flow) is physiologically tightly autoregulated, and variations in systemic blood pressure are generally not transmitted to these capillaries. If the BBB is disrupted, transcapillary water transport will be determined by the differences in hydrostatic and colloid osmotic pressure between the intra- and extracapillary compartments. Under these pathological conditions, pressure autoregulation of cerebral blood flow is likely to be impaired and intracapillary hydrostatic pressure will depend on variations in systemic blood pressure. The volume-targeted 'Lund concept' can be summarized under four headings: (1) Reduction of stress response and cerebral energy metabolism; (2) reduction of capillary hydrostatic pressure; (3) maintenance of colloid osmotic pressure and control of fluid balance; and (4) reduction of cerebral blood volume. The efficacy of the protocol has been evaluated in experimental and clinical studies regarding the physiological and biochemical (utilizing intracerebral microdialysis) effects, and the clinical experiences have been favorable.

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.

[Current alternative therapy of post-traumatic brain edema]. / Ny alternativ terapi av posttraumatiskt hjärnödem.

Mortality is high among patients developing post-traumatic brain oedema and increased intracranial pressure following severe head injury. Although routine treatment varies from one centre to another it often includes one or more of such measures as hyperventilation, high-dose barbiturate therapy, osmotherapy or the drainage of cerebrospinal fluid. The preservation of high cerebral perfusion pressure is fundamental to traditional treatment, often combined with inotropic support, as ischemia is considered to be a crucial factor with regard to the development of secondary injuries and oedema in the brain. In the article is described a new treatment of posttraumatic brain oedema, based on the hypothesis that the development of oedema is instead largely due to disturbance of brain volume control mechanisms resulting from increased permeability of the semi-permeable blood-brain barrier. If this hypothesis is true, oedema therapy should include measures to decrease hydrostatic capillary pressure and preserve normal colloid osmotic pressure. Our new therapy therefore includes hypotensive treatment in the form of beta 1-blockade and alpha 2-stimulation and precapillary vasoconstriction by dihydroergotamine (DHE) infusion, all of which reduce capillary pressure. The DHE may also constrict venous vessels, resulting in reduced blood volume. Colloid osmotic pressure is preserved with albumin infusion. Normovolemia is attained despite manifest fluid balance. With this therapy both mortality and morbidity have been reduced significantly, as compared to retrospective figures for a comparable control group.

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.