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Biomedical subjects

J Mikkelsen

Publications and source records attributed to J Mikkelsen.

At least 19 recordsLinked to original sources

Hydrodynamic drag of diving birds: effects of body size, body shape and feathers at steady speeds.

For birds diving to depths where pressure has mostly reduced the buoyancy of air spaces, hydrodynamic drag is the main mechanical cost of steady swimming. Drag is strongly affected by body size and shape, so such differences among species should affect energy costs. Because flow around the body is complicated by the roughness and vibration of feathers, feathers must be considered in evaluating the effects of size and shape on drag. We investigated the effects of size, shape and feathers on the drag of avian divers ranging from wing-propelled auklets weighing 75 g to foot-propelled eiders weighing up to 2060 g. Laser scanning of body surfaces yielded digitized shapes that were averaged over several specimens per species and then used by a milling machine to cut foam models. These models were fitted with casts of the bill area, and their drag was compared with that of frozen specimens. Because of the roughness and vibration of the feathers, the drag of the frozen birds was 2-6 times that of the models. Plots of drag coefficient (C(D)) versus Reynolds number (Re) differed between the model and the frozen birds, with the pattern of difference varying with body shape. Thus, the drag of cast models or similar featherless shapes can differ both quantitatively and qualitatively from that of real birds. On the basis of a new towing method with no posts or stings that alter flow or angles of attack, the dimensionless C(D)/Re curves differed among a size gradient of five auklet species (75-100g) with similar shapes. Thus, extrapolation of C(D)/Re curves among related species must be performed with caution. At lower speeds, the C(D) at a given Re was generally higher for long-necked birds that swim with their neck extended (cormorants, grebes, some ducks) than for birds that swim with their head retracted (penguins, alcids), but this trend was reversed at high speeds. Because swimming birds actually travel at a range of instantaneous speeds during oscillatory strokes, species variations in drag at different speeds must be considered in the context of accelerational stroking.

Animals↗

Vacuum assisted venous drainage (VAVD).

Poor venous drainage is a common problem in cardiac surgery, causing trouble for the surgeon and adverse effects to the patient. Smaller incisions for minimally invasive cardiac surgery require smaller venous catheters. In this study the function, safety and possible benefits of a system for vacuum assisted venous drainage has been tested experimentally and applied clinically. A vacuum regulator ('The Hamlet box') and safety procedures were developed. The system was characterized in vitro in regard to the relationship between vacuum, catheter size, and blood temperature and flow. The clinical study included 54 adult patients, coronary artery bypass graft surgery and valve operations. Venous cannulation was bi-caval with two 24 Fr catheters. All the perfusions were essentially event free, and the system was easy to manage and regulate. Venous drainage was totally adequate, irrespective of the position of the heart, and less fluid was added during the perfusions (a median of 250 ml/patient compared to a median of 1000 ml/patient in the control group). There has been no evidence of increased haemolysis or other adverse effects. All patients were hospital survivors and had uneventful postoperative courses. Vacuum assisted venous drainage is now used routinely, and further studies are under way to develop the system and clarify the physiological effects.

Cardiac Surgical Procedures↗

Engineering stability of the insulin monomer fold with application to structure-activity relationships.

To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we have analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably described on the basis of a two-state denaturation scheme; however, the observation of subtle differences between near- and far-UV CD detected unfolding indicates that intermediates may be present. Three regions of the insulin molecule are analyzed in detail with respect to their contribution to folding stability, i.e., the central B-chain helix, the NH2-terminal A-chain helix, and the B25-B30 extended chain region. Considerable enhancement of folding stability is engineered by mutations at the N-cap of the central B-chain helix and at the C-cap of the NH2-terminal A-chain helix. Mutations that confer increased stability in these regions are identical to those that lead to enhanced biological activity. In contrast, for insulin species modified in the B25-B30 region of the molecule, we observe no correlation between global folding stability and bioactivity. Mutations in the three regions examined are found to affect stability in a nearly independent fashion, and stabilizing mutations are generally found to enhance the cooperativity of the unfolding transition. We conclude that highly potent insulins (i.e., HisA8, ArgA8, GluB10, and AspB10) elicit enhanced activity because these mutations stabilize structural motifs of critical importance for receptor recognition.

Amino Acid Sequence↗

[Ambulatory surgery and anesthesia. An inquiry study].

Patients who had undergone surgical procedures as out-patients in either general or local anaesthesia were asked to complete a questionnaire with the aim of evaluating how they had experienced their operation, especially with respect to treatment and prevention of postoperative pain. Over a 12 month period, 851 patients (251 men and 600 women) who had undergone either abdominal, orthopaedic or gynaecological surgery as out-patients were given the questionnaire. Five hundred and fifteen patients (166 men and 349 women), i.e. 61%, answered. Nineteen percent had had their operation performed in local or regional anaesthesia, 30% in a combination of general anaesthesia with local infiltration, the remaining patients were operated under general anaesthesia. Eighty to ninety percent were satisfied with the preoperative information, and 88% were satisfied with the anaesthesia. Sixty-nine percent had had either no pain or almost no pain within the first 24 hours after the operation. Six percent found it difficult to stay home. Sixteen percent needed some kind of medical contact. Ninety-two percent said they would prefer out-patient surgery again as opposed to hospitalization, were they to need another operation. In conclusion, we found that these types of out-patient operations were acceptable to most of our patients (92%). We recommend widespread use of local anaesthetics in combination with general anaesthesia in order to minimize postoperative pain and facilitate the effect of postoperative analgetics.

Ambulatory Surgical Procedures↗

Heterogeneity in the tyrosine sulfation of Chinese hamster ovary cell produced recombinant FVIII.

By the use of recombinant technology, several stable Chinese hamster ovary (CHO) cell lines expressing human FVIII were established. Thrombin treatment and SDS-PAGE analysis of the purified recombinant FVIII (rFVIII) revealed a striking difference from plasma-derived FVIII (pFVIII). A 43-kDa fragment of the FVIII heavy chain appears as a double band from rFVIII, while a single band from pFVIII is observed. All other fragments from the two samples appeared similar by SDS-PAGE. The heterogeneity is caused by incomplete tyrosine sulfation of one or more of the three potential tyrosine sulfation sites (Tyr718, Tyr719, Tyr723). To investigate if there is a general limitation and heterogeneity in the tyrosine sulfation of rFVIII, two other potential tyrosine sulfation sites on the FVIII light chain (Tyr1664, Tyr1680) were analyzed. The results show that both sites on the pFVIII light chain and on the rFVIII light chain are completely sulfated. The limitation of CHO cells to tyrosine sulfate rFVIII is therefore only restricted to a few sites. The two sulfated forms of rFVIII can easily be separated by ion-exchange chromatography, indicating the importance of the sulfate groups on the charge and/or conformation of FVIII. Both forms of rFVIII possess identical in vitro coagulation activity, von Willebrand factor binding, and thrombin activation profile. However, the difference in tyrosine sulfation may change other biological properties of FVIII.

Animals↗

Molecular cloning and sequencing of a cDNA encoding the thioesterase domain of the rat fatty acid synthetase.

A cloned cDNA containing the entire coding sequence for the long-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase I) component as well as the 3'-noncoding region of the fatty acid synthetase has been isolated using an expression vector and domain-specific antibodies. The coding region was assigned to the thioesterase I domain by identification of sequences coding for characterized peptide fragments, amino-terminal analysis of the isolated thioesterase I domain and the presence of the serine esterase active-site sequence motif. The thioesterase I domain is 306 amino acids long with a calculated molecular mass of 33,476 daltons; its DNA is flanked at the 5'-end by a region coding for the acyl carrier protein domain and at the 3'-end by a 1,537-base pairs-long noncoding sequence with a poly(A) tail. The thioesterase I domain exhibits a low, albeit discernible, homology with the discrete medium-chain S-acyl fatty acid synthetase thioester hydrolases (thioesterase II) from rat mammary gland and duck uropygial gland, suggesting a distant but common evolutionary ancestry for these proteins.

Amino Acid Sequence↗

Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution.

Using the tyrosine fluorescence quenching as a criterion for acyl-CoA binding, we have shown that acyl-CoA-binding protein (ACBP) binds acyl-CoA esters with a chain length greater than C8 with equal affinity. The binding studies indicated a binding stoichiometry of 1 mol of acyl-CoA/2 mol of ACBP. The protein was found in liver, adipose tissue, intestinal mucosa, kidney, heart, brain, muscles and mammary gland. The highest concentration was found in liver cytosol and the lowest in muscles and mammary gland. ACBP could not be shown to bind non-esterified fatty acids.

Acyl Coenzyme A↗

Amino acid sequence of acyl-CoA-binding protein from cow liver.

Acyl-CoA-binding protein from bovine liver was purified with the use of reverse-phase h.p.l.c. in the final step. The complete amino acid sequence was determined by using a combination of gas-phase Edman degradation and electron-impact and fast-atom-bombardment mass spectrometry. The sequence was confirmed by determination of the Mr by plasma-desorption time-of-flight mass spectrometry.

Amino Acid Sequence↗

Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase.

Cloned cDNAs containing coding sequences for domains proximal to the carboxy terminus of the rat fatty acid synthetase have been isolated using an expression vector and domain-specific antibodies. The coding regions were assigned to specific domains of the multifunctional complex by identification of sequences coding for characterized peptide fragments and by recognition of sequences homologous to other monofunctional enzymes. Two clones contain the entire coding region for the acyl carrier protein domain. The sequence is flanked at the 3'-end by a region coding for the thioesterase domain and at the 5'-end by a sequence coding for a reductase, most likely the ketoreductase domain. Thus the ordering of these domain-coding regions in the fatty acid synthetase mRNA is established. The acyl carrier protein domain exhibits about 25% homology with that of the discrete monofunctional acyl carrier proteins of Escherichia coli, spinach and barley, the ketoreductase domain exhibits about 25% homology with bacterial dihydrofolate reductases and the active site of the thioesterase domain exhibits both primary and secondary structural features common to the serine proteases. These findings lend support to the hypothesis that the polyfunctional fatty acid synthetase probably arose by a complex evolutionary process involving fusion of genes coding for seven individual enzymes.

Acyl Carrier Protein↗

A novel procedure for the preparation and characterization of catalytically active fatty acid synthetase immobilized on sepharose beads.

A novel procedure for immobilization of enzymatically active fatty acid synthetase is presented. The enzyme is coupled to a Sepharose 4B matrix containing covalently attached antibodies which recognize, and bind specifically to, the thioesterase domain of this polyfunctional enzyme. A continuous flow system is described for assay of the immobilized enzyme. Fatty acid synthetase activity apparently is not limited by movement of substrates through the Nernst diffusion layer surrounding the matrix particles, since normal Michaelis-Menten kinetics are observed and reaction rates are independent of flow rate. The Km values for acetyl-CoA and malonyl-CoA, the pH/activity profile, and the reaction products are essentially the same as for the freely soluble enzyme, although the specific activity is lower by about 55%. The preparation and characterization of immobilized subunits of the enzyme could provide a valuable approach for studying the role of structural and functional subunit interactions in the enzyme. In addition, the immobilized enzyme offers a model for studying the properties of this enzyme in a highly structured environment such as might exist in vivo, permitting study of both physical and functional interactions of fatty acid synthetase with other lipogenic enzymes.

Acetyl Coenzyme A↗

Interaction of rat mammary gland thioesterase II with fatty acid synthetase is dependent on the presence of acyl chains on the synthetase.

The interaction between rat mammary gland thioesterase II and fatty acid synthetase has been studied by a variety of physicochemical techniques. Pyrene-labeled thioesterase II does not exhibit increased fluorescence anisotropy when mixed with fatty acid synthetase, suggesting that the enzymes do not readily form a complex. Nevertheless, the functional interaction between the enzymes can be easily demonstrated by observing the hydrolysis, by unmodified thioesterase II, of acyl chains from their thioester linkage to the 4-phosphopantetheine of the fatty acid synthetase. This hydrolytic reaction is not inhibited even in the presence of a large excess of fatty acid synthetase with vacant 4'-phosphopantetheine thiols, indicating that interaction occurs only between thioesterase and fatty acid synthetase species which carry acyl chains on the 4'-phosphopantetheine thiols. A novel model system was devised which allowed us to explore the nature of the physical interaction between the two enzymes under conditions where the synthetase was actively engaged in acyl chain assembly. Fatty acid synthetase was treated with phenylmethanesulfonyl fluoride to inhibit its resident thioesterase activity, immobilized via a specific antibody to a column of Sepharose 4B, and exposed to the substrates required for acyl-enzyme assembly. When thioesterase II was introduced to the column, it passed through unretarded even though it efficiently catalyzed hydrolysis of the immobilized S-acyl synthetase en route. These results indicate that the two enzymes associate when an acyl chain is present on the synthetase and that they dissociate rapidly following completion of the catalytic process. Thus, the mammary system differs from that of the avian uropygial gland in which the two enzymes associate to form a stable complex even in the absence of substrates.

Animals↗

Stoichiometry of substrate binding to rat liver fatty acid synthetase.

Two rat liver fatty acid synthetase preparations, containing 1.6 and 2.0 mol of 4'-phosphopantetheine/mol of synthetase, showed specific activity of 2006 and 2140 nmol of NADPH oxidized/min per mg of protein respectively. The two synthetase preparations could be loaded with either 3.3-4.4 mol of [1-14] acetate or 2.9-3.7 mol of [2-14C]malonate, by incubation with either [1-14C] acetyl-CoA or [2-14C]malonyl-CoA. The 4'-phosphopantetheine site could be more than 90% saturated and the serine site about 80% saturated with malonate derived from malonyl-CoA. However, with acetyl-CoA as substrate, binding at both the 4'-phosphopantetheine and cysteine thiol sites did not reach saturation. We interpret these results to indicate that, whereas the equilibrium constant for transfer of substrates between the serine loading site and the 4'-phosphopantetheine site is close to unity, that for transfer of acetyl moieties between the 4'-phosphopantetheine and cysteine sites favours formation of the 4'-phosphopantetheine thioester. Thus, despite the apparent sub-stoichiometric binding of acetate, the results are consistent with a functionally symmetrical model for the fatty acid synthetase which permits simultaneous substrate binding at two separate active centres.

Acetates↗

Evidence that the medium-chain acyltransferase of lactating-goat mammary-gland fatty acid synthetase is identical with the acetyl/malonyltransferase.

Competitive binding experiments with malonyl-CoA and [1-14C]acetyl-CoA, [1-14C]butyryl-CoA or [1-14C]decanoyl-CoA indicate that all these substrates are transferred to lactating-goat mammary-gland fatty acid synthetase by the same transferase. Isolation and determination of the amino acid sequence of [1-14C]decanoyl-labelled CNBr-cleavage peptide from the decanoyltransferase site showed that this transferase is identical with the acetyl/malonyltransferase.

Acetyl Coenzyme A↗