Biomedical subjects
Publications and source records attributed to L K Nielsen.
H(+)-ATPase is considered essential for growth of Lactococcus lactis. However, media containing hemin restored the aerobic growth of an H(+)-ATPase-negative mutant, suggesting that hemin complements proton extrusion. We show that inverted membrane vesicles prepared from hemin-grown L. lactis cells are capable of coupling NADH oxidation to proton translocation.
Explore the source record for details and available documents.
The lag-burst phenomenon in the phospholipase A(2) mediated hydrolysis of phospholipid bilayers is for the first time demonstrated in an atomic force microscopy (AFM) study. Simultaneous AFM measurements of the degree of bilayer degradation and the physical-chemical state of the membrane reveals growing nanoscale indentations in the membrane during the lag phase. It is argued that these indentations are domains of hydrolysis products (lysoPC/PC) which eventually trigger the burst. The rate of the rapid hydrolysis following the burst is found to be proportional to the length of the edge between membrane adsorbed and desorbed to the mica base. The observed maximal rate of membrane degradation is approx. 0.2 mmol lipid/min/mol lipase in solution.
A stoichiometric model of Clostridium acetobutylicum and related strains has been previously derived. The stoichiometric matrix of the model contains a singularity which has prevented the calculation of a unique set of fluxes which describe the primary metabolic activity. To resolve the singularity, we have developed a non-linear constraint relating the acetate and butyrate uptake fluxes. Subsequently, we developed a software package utilizing a model independent heuristic global optimization approach to solve the resultant non-linear problem. We have validated the use of the non-linear constraint by correlating calculated butyrate production pathway flux profiles with measured intracellular pH profiles. Finally, we examined a controlled batch fermentation to determine that the acid formation pathways play critical roles throughout solventogenesis. The broader usefulness of reformulating the stoichiometric model as a constrained minimization problem is discussed.
Hematopoietic cell culture, or ex vivo expansion of hematopoietic cells, is an enabling technology with many potential applications in bone-marrow transplantation, immunotherapy, gene therapy, and the production of blood products. Hematopoietic cultures are complex, with many different cell types of different stages of development present at any given point in time and never in steady state. Moreover, these cells interact strongly with each other and the environment through cytokines (growth factors) and adhesion molecules, as well as through their metabolism. Despite these significant challenges, cell products produced in bioreactors have shown promise in recent phase 1 clinical trials.
The results of studies using suppressive doses of L-T4 on benign solitary solid cold thyroid nodules have been conflicting. Recently, intranodular injection of absolute ethanol has been proposed as an effective treatment, but has been evaluated only in uncontrolled studies. Our objective was to evaluate the effect of two alternative medical treatment modalities, percutaneous ethanol injection therapy and L-T4, on the benign solitary solid cold thyroid nodule. In a prospective randomized clinical trial, 50 euthyroid patients with a single solid colloid thyroid nodule causing local discomfort were assigned to a single intranodular injection of sterile 98% ethanol (n = 25) or suppressive doses of L-T4 (n = 25). We aimed at an ethanol dose of 20-50% of the pretreatment nodular volume. The initial daily dose of L-T4 was 1.5 microg/kg BW and was adjusted monthly during the first 6 months to reduce serum TSH to subnormal levels (<0.40 mU/L). Thyroid nodule volume and total thyroid volume were assessed by ultrasound, and thyroid function was determined by routine assays before and during follow-up. Symptom scores before and at 12 months were evaluated by a questionnaire rating pressure symptoms and cosmetic symptoms. The median ethanol dose given was 21% [95% confidence interval (CI), 18;25] of the pretreatment nodule volume. In this group, the median reduction in nodule volume was 47% (CI, 33;57; P < 0.0001) compared to 9% (CI, -7;22; P = 0.09) in the L-T4 group. The difference between the two treatment regimens was statistically significant (P < 0.0001). The median reduction in perinodular thyroid volume was 20% (CI, 11;31; P = 0.03) in the L-T4 group, whereas no change was seen in the ethanol group (-2.5%; CI, -18;11; P = 0.9). Fourteen of 25 (56%) patients treated with ethanol injection and 8 of 25 (32%) treated with L-T4 had complete relief of symptoms at 12 months of follow-up (P = 0.09). No major side-effects were seen in either group. Percutaneous ethanol injection therapy administered as a single small dose results in a satisfactory clinical response in approximately 50% of patients by halving the nodule volume. The thyroid nodule-reducing effect of L-T4 suppressive therapy is insignificant, but a subjective satisfactory clinical response is seen in a subgroup of patients, probably explained by the concomitant reduction of perinodular thyroid volume.
Explore the source record for details and available documents.
We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage.
A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively.
Explore the source record for details and available documents.
Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor. The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density. For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place. It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed. We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection. Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle. No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection.
UNLABELLED: Polymorphonuclear neutrophil (PMN) stimulation and degranulation can be mediated by the cytokines and by complement activation. The aim of the present study was to measure TNF alpha, IL-1 alpha, IL-6 and C3d in relation to postoperative increase in lactoferrin and elastase alpha-1-proteinase inhibitor (E alpha-1-PI) levels. Eleven patients undergoing thoracic surgery took part in the study. Blood leucocytes, E alpha-1-PI, lactoferrin and C3d were measured preoperatively, at the end of surgery and postoperatively, at 4 h and on day 1, 2, 3 and 5. TNF alpha, IL-1 alpha and IL-6 were measured preoperatively, at the end of surgery and postoperatively, at 4 h, and on days 1 and 5. The leucocyte count, lactoferrin and E alpha-1-PI levels increased significantly postoperatively (P < 0.01). There was no significant change in C3d values. Plasma IL-6 levels were unchanged in the postoperative period. Plasma TNF alpha and IL-1 alpha were detectable at low levels in only two and four patients, respectively. CONCLUSION: The postoperative increase in blood levels of PMN lactoferrin and E alpha-1-PI complexes observed in the present study was not accompanied by complement activation, or increased blood levels of IL-6.
Explore the source record for details and available documents.
Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002-0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4 x 10(9) cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.
The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was found to be inconsistent. A superior estimator based on the maximum likelihood principle was developed. The results are discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.
We have studied the effect of dietary supplementation with 4 g of n-3 polyunsaturated fatty acids (n-3 PUFA) daily for 6 wk on plasma lipids, haemostasis and monocyte chemotaxis in 10 patients with untreated hypertension. Total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides did not change, but the ratio of total to HDL-cholesterol was significantly reduced after the fish oil supplement. Platelet function was unaltered by intake of n-3. Plasma fibrinogen and fibronectin decreased after supplementation with n-3 PUFA, while the effects on fibrinolysis were equivocal. Monocyte chemotaxis was reduced by the supplement. These data lend support to a role for an increased intake of n-3 PUFA in the management of patients with hypertension.
In 10 patients admitted to hospital with diabetic ketoacidosis plasma prostanoids 6-keto-PGF alpha, thromboxane B2 and PGE2 were studied before treatment and following recovery. During ketoacidosis the median plasma 6-keto-PGF1 alpha and PGE2 were significantly increased compared to those of a normal reference group: 5.2 pg/ml and 3.9 pg/ml versus 1.7 pg/ml and 0.4 pg/ml (p less than 0.01 and p less than 0.05). In response to therapy both prostanoids decreased significantly towards a normal level, 6-keto-PGF1 alpha: 0.5 pg/ml p less than 0.01 and PGE2: 0.08 p less than 0.05 respectively. The changes in plasma 6-keto-PGF1 alpha were negatively correlated to changes in pH, rho: -0.7788 p = 0.0135, whereas the changes in PGE2 were positively correlated to serum creatinine at admittance, rho: 0.6976, p = 0.0368 and to the amount of intravenous fluid and insulin used during treatment, rho: 0.7500 p = 0.0126 and rho: 0.8424, p = 0.0023 respectively. Plasma thromboxane B2 concentrations were not elevated and did not change after treatment of the ketoacidosis.
A latex-agglutination test (Rapi-Tex) was used for semiquantitative estimation of myoglobin concentration in 89 serum samples obtained from ten patients undergoing lower-limb arterial embolectomy. Each serum sample was tested with Rapi-Tex both undiluted and after 1:50 dilution with saline. The myoglobin concentration was additionally measured with enzyme-immunoassay. In the undiluted samples there were five 'false negative' Rapi-Tex results--two in the myoglobin concentration interval 0-300 micrograms and three in the samples with myoglobin exceeding 25,000 micrograms/l--and four 'false positive' results. None of the diluted samples gave rise to false classification. Using 1:50 diluted serum, the negative predictive value of the Rapi-Tex test for identification of a myoglobin concentration greater than 5,000 micrograms/l was 0.99 (95% confidence limits 0.94-1.00), and the positive predictive value was 1.00 (0.61-1.00). Rapi-tex is concluded to be a useful emergency test for severe hypermyoglobinemia.