Biomedical subjects

J Nielsen

Publications and source records attributed to J Nielsen.

At least 73 records · Page 4Linked to original sources

PubMed2001

Metabolic engineering of the morphology of Aspergillus.

The morphology of filamentous organisms in submerged cultivation is a subject of considerable interest, notably due to the influence of morphology on process productivity. The relationship between process parameters and morphology is complex: the interactions between process variables, productivity, rheology, and macro- and micro-morphology create difficulties in defining and separating cause and effect. Additionally, organism physiology contributes a further level of complexity which means that the desired morphology (for optimum process performance and productivity) is likely to be process specific. However, a number of studies with increasingly powerful image analysis systems have yielded valuable information on what these desirable morphologies are likely to be. In parallel, studies on a variety of morphological mutants means that information on the genes involved in morphology is beginning to emerge. Indeed, we are now beginning to understand how morphology may be controlled at the molecular level. Coupling this knowledge with the tools of molecular biology means that it is now possible to design and engineer the morphology of organisms for specific bioprocesses. Tailor making strains with defined morphologies represents a clear advantage in optimization of submerged bioprocesses with filamentous organisms.

Aspergillus↗

PubMed2001

Patients with the major and minor form of hyperekplexia differ with regards to disynaptic reciprocal inhibition between ankle flexor and extensor muscles.

The aim of the present study was to investigate the contribution of reciprocal inhibition to muscle tone by examining the transmission in the reciprocal inhibitory pathway in patients with a known defect in the glycine receptor. The study was performed in eight patients with hereditary hyperekplexia, six with the major form and two with the minor form of the disease. A mutation in the alpha1-subunit of the glycine receptor had been demonstrated in the patients with the major form, whereas no mutation was seen in the patients with the minor form. Disynaptic reciprocal inhibition, which is presumed to be mediated by glycine, was not seen in the patients with the major form of the disease, while it could be evoked in the patients with the minor form of the disease. Presynaptic inhibition, which is presumed to be mediated by GABA, was seen in both types of patients. It is concluded that the major form of hereditary hyperekplexia is associated with impaired transmission in glycinergic reciprocal inhibitory pathways. The findings demonstrate the importance of reciprocal inhibition for the muscle tone in man, and it is suggested that the impaired reciprocal inhibition seen in patients with a defect in the glycine receptor may contribute to the increased muscle stiffness that is observed in these patients.

Adult↗

PubMed2001

alpha-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans.

The effect of biomass concentration on the formation of Aspergillus oryzae alpha-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of alpha-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2-12 g dry weight kg(-1). When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzae alpha-amylase gene (creA) was deleted, no significant decrease in the specific rate of alpha-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific alpha-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium.

Aspergillus nidulans↗

PubMed2001

Metabolic engineering.

Metabolic engineering has developed as a very powerful approach to optimising industrial fermentation processes through the introduction of directed genetic changes using recombinant DNA technology. Successful metabolic engineering starts with a careful analysis of cellular function; based on the results of this analysis, an improved strain is designed and subsequently constructed by genetic engineering. In recent years some very powerful tools have been developed, both for analysing cellular function and for introducing directed genetic changes. In this paper, some of these tools are reviewed and many examples of metabolic engineering are presented to illustrate the power of the technology. The examples are categorised according to the approach taken or the aim: (1) heterologous protein production, (2) extension of substrate range, (3) pathways leading to new products, (4) pathways for degradation of xenobiotics, (5) improvement of overall cellular physiology, (6) elimination or reduction of by-product formation, and (7) improvement of yield or productivity.

Biotechnology↗

PubMed2001

Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture.

The central carbon metabolism of the nystatin-producing strain Streptomyces noursei ATCC 11455 was evaluated by 13C-labelling experiments. A batch fermentation was examined during the idiophase by GC-MS measurements of the labelling patterns of amino acids in the biomass. The labelling patterns of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation.

Amino Acids↗

PubMed2001

Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration.

With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factors-that needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.

Biomass↗

PubMed2001

Production of a heterologous proteinase A by Saccharomyces kluyveri.

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.

Aspartic Acid Endopeptidases↗

PubMed2001

Influence of carbon source on alpha-amylase production by Aspergillus oryzae.

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.

Aspergillus oryzae↗

PubMed2001

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production.

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this process therefore represents a new route for the production of 7-ADCA, which serves as a precursor for the production of many semi-synthetic cephalosporins. In this study, one low- and one high-yielding strains were characterised and the specific productivities of ad-7-ADCA and byproducts of the biosynthetic pathway were compared. The fluxes through the biosynthetic pathway were quantified and it was found that there was a 30% higher flux through the expandase in the high-yielding strain. In both strains, there was a significant degradation of adipate. Furthermore, the initial adipate concentration in batch cultures was shown to have a positive effect on the formation of ad-7-ADCA.

Adipates↗

PubMed2001

Evaluation of disease-state management of dialysis patients.

Dialysis patients are the only Medicare beneficiaries prohibited from joining managed care plans. Concerns have been raised about the ability of such plans to provide the comprehensive care required by patients with this complex condition. However, more than 20,000 dialysis patients belong to such plans because they were enrolled before developing end-stage renal disease (ESRD). Disease-state management, successfully applied to patients with diabetes mellitus and congestive heart failure, is now being used in patients with ESRD. Standardized mortality ratios (SMRs) and standardized hospitalization ratios (SHRs) were calculated for 1998 and 1999 in 1,541 patients enrolled in the RMS Disease Management program of renal disease-state management using US Renal Data System methods. SMRs were 0.643 and 0.806 for 1998 and 1999, respectively, significantly different from 1.0 for both years (P < 0.001). SHRs were 0.620 and 0.503 for 1998 and 1999, respectively, significantly different from 1.0 for both years (P < 0.001). Although additional studies are needed to define the aspects of care that are most important for the outcomes seen, this study shows that favorable outcomes are achievable for this vulnerable patient population within a managed care setting that applies coordinated approaches to care.

Adolescent↗

PubMed2001

A prospective study of the relationship between exposure and specific antibodies in workers exposed to organic acid anhydrides.

BACKGROUND: The exposure-response relationships for the induction of specific IgE and IgG were evaluated in a prospective study of workers exposed to organic acid anhydrides (OAAs). Special attention was paid to the modifying effects of atopy and smoking. METHODS: The subjects were 163 previously unexposed persons exposed to epoxy resins with hexahydro-, methylhexahydro-, and methyltetrahydrophthalic anhydride as curing agents. The levels of OAAs in air and of specific IgE and IgG in serum were recurrently monitored. The mean observation time was 32 (1-105) months. RESULTS: The mean combined OAA exposure of the subjects was 15.4 (< 1-189) microg/m3. Positive specific IgE was demonstrated by 21 (13%) subjects with a mean induction time of 8.8 (1-35) months. The incidence of sensitization was 4.1 cases/1000 months at risk. The relative risk (OR) for atopics was 5.4 (1.9-15.3; 95% CI). An exposure-response relationship was demonstrated by an increasing risk of sensitization with increasing exposure. CONCLUSION: An association between exposure and atopy, respectively, and the induction of specific antibodies against OAAs were observed. The risk for atopics was comparable with the risk for the subjects in the most exposed group.

Adolescent↗

PubMed2001

Thymus size and head circumference at birth and the development of allergic diseases.

BACKGROUND: The positive association between a large head circumference at birth and total serum IgE levels has been suggested to be due to negative associations between head circumference at birth and thymus development and between thymus development and total serum IgE levels. OBJECTIVES: To examine the associations between head circumference and thymus size at birth and the development of allergic disease. METHODS: The size of the thymus was assessed by sonography during the first week of life in 149 healthy term infants. Information on birth characteristics and mode of delivery was collected at delivery. The presence of allergic disease was assessed 5 years later by mailed questionnaires, which were returned by 85% of the eligible families. RESULTS: At birth, head circumference was positively associated with thymus size (P < 0.001). In all, 27 (23%) of the children had developed at least one allergic disease. Multivariate analysis revealed that both parental allergy (Prevalence Ratio and 95% CI) = 3.18 (1.49-6.78)) and caesarean delivery (2.62 (1.48-4.64)) were independently correlated with allergic disease, whereas thymus size was not. CONCLUSIONS: Our study does not support that a large head circumference is associated with a small thymus size, nor that a small thymus size is associated with allergic disease. Whether thymus size at birth is related to total serum IgE levels still remains to be elucidated.

Birth Weight↗

PubMed2001

The probiotic potential against vibriosis of the indigenous microflora of rainbow trout.

The antibacterial properties of the indigenous microflora of rainbow trout (Oncorhynchus mykiss Walbaum) and the potential use of inhibitory bacteria as fish probiotics were investigated. A total of 1018 bacteria and yeasts were isolated on tryptone soy agar (TSA) from skin, gills and intestine. Forty-five of these inhibited growth of the fish pathogenic bacterium Vibrio anguillarum in a well diffusion assay. The antagonism was most prominent among Pseudomonas spp., as 28 (66%) of the antagonistic bacteria belonged to this genus, despite constituting only 15% of the total tested flora. As pseudomonads are typically siderophore producers, chrome azurol S (CAS) agar was used as a semi-selective medium for isolation of antagonistic bacteria. On this medium, 75% of the iron-chelating strains were inhibitory to V. anguillarum. Eight strains out of a subset of 11 antagonists caused a 3-6 log unit reduction in the density of V. anguillarum [measured by polymerase chain reaction (PCR) detection in a most probable number (MPN) regimen] in a broth co-culture assay. Survival of rainbow trout infected with vibriosis was improved 13-43% by six out of nine antagonistic strains tested in vivo. All disease-protecting strains were pseudomonads, isolated from CAS plates, whereas two Carnobacterium spp. that were antagonistic in in vitro well diffusion assays did not alter the accumulated mortality of rainbow trout. The addition of live bacterial cultures to fish-rearing water may thus improve survival of the fish; however, in vitro antagonism could not completely predict an in vivo effect. Further studies on the underlying mechanism of activity are required to design appropriate selection criteria for fish probiotic bacteria.

Animals↗

PubMed2001

Spatial summation of heat pain in males and females.

Sex differences in pain sensitivity have been found to vary between considerable and negligible. It has appeared that the pain stimulation method is critical in this context. It was assumed this might be due to the different degrees of spatial summation associated with the different pain stimulus modalities. Hence, sex differences were investigated in spatial summation of heat pain in 20 healthy women and 20 healthy men of similar age. Pain thresholds were assessed by a tracking procedure and responses to supra-threshold pain stimulation by numerical ratings. Heat stimuli were administered by a thermode with contact areas of 1, 3, 6 and 10 cm2. Pain thresholds were significantly higher with smaller areas stimulated than with larger ones. No significant effect of area was found for the ratings of the supra-threshold stimuli, the intensities of which were tailored to the individual pain threshold. Consequently, spatial summation of heat pain appeared to result mainly in a shift of the pain threshold on the ordinate and not a change of slope of the stimulus-response function in the pain range. In neither of the two pain parameters were there any sex differences. Therefore, the present study demonstrated that sex differences in spatial summation of heat pain are unlikely.

Female↗

PubMed2001

Morphological characterization of Aspergillus nidulans: growth, septation and fragmentation.

The influence of the sepA gene on the growth of Aspergillus nidulans has been investigated by characterizing and comparing the parental strain A28 (pabaA6 biA1) with the sepA null mutant (sepA4DeltaBM:). The sepA gene is known to affect the septation process in A. nidulans, therefore the sepA4DeltaBM: strain does not produce any septa during the first hours of growth. During batch cultivations sepA4DeltaBM: shows an abrupt decrease in specific growth rate and more pronounced fragmentation (in response to elevated stirrer speed) than the parental strain. Higher specific fragmentation rates (q(frag)) were obtained for the sepA4DeltaBM: strain. The physiological reasons for the differences have been investigated by employing fluorescent stains. Computerized image analysis revealed that the more pronounced fragmentation in the mutant was due to the lower number and irregular spacing of septa (visualized by calcofluor white staining), which resulted in a weaker hyphal structure that is more vulnerable to shear stress and fragmentation than the parental strain. This led to a loss of active biomass (determined by Mag fura staining) from the hyphae of the mutant, which had failed to compartmentalize by formation of septa, in turn resulting in decreased specific growth rates for the culture.

Aspergillus nidulans↗

PubMed2001

The impact of GAL6, GAL80, and MIG1 on glucose control of the GAL system in Saccharomyces cerevisiae.

The role of the proteins encoded by the GAL80 gene, the MIG1 gene and the GAL6 gene in glucose control of galactose consumption by Saccharomyces cerevisiae was studied by physiological characterisation of various GAL mutant strains. Dynamic experiments with the CEN.PK 113-7D wild-type strain and a deltagal80deltamig1 double-mutant strain in aerobic nitrogen-limited continuous cultivations at a dilution rate of 0.1 h(-1), showed simultaneous glucose and galactose consumption by the deltagal80deltamig1 strain. The wild-type strain did not consume galactose in the presence of glucose. Aerobic batch cultivations on glucose-galactose mixtures with the wild-type strain and with recombinant strains with a de-regulated GAL system (the deltagal80deltamig1 strain, a deltagal6 deleted strain, a deltagal6deltagal80deltamig1 triple mutant, and a deltagal6deltagal80deltamig1 triple mutant harbouring a GAL4 high-copy vector) were carried out. Generally, a reduction of glucose control lowered the maximum specific growth rate on glucose and increased the ethanol yield obtained on galactose with more than 100%. In contrast to the wild-type strain, the deltagal6deltagal80deltamig1 triple mutant strain consumed glucose and galactose simultaneously, and this strain also showed the highest ethanol production with an overall ethanol yield of 0.35 g g-1 sugar, which is 17% higher than the yield on glucose obtained with the wild-type strain. GAL80 and MIG1 were demonstrated to be responsible for the majority of the glucose control on the GAL system, whereas GAL6 has a minor role in glucose control. Deletion of GAL6 was shown to have a major impact on biomass and ethanol formation when cells were grown on galactose, and from the data obtained we speculate that Gal6 may be involved in mRNA degradation of the GAL gene transcripts.

Culture Media↗

PubMed2001

Network identification and flux quantification in the central metabolism of Saccharomyces cerevisiae under different conditions of glucose repression.

The network structure and the metabolic fluxes in central carbon metabolism were characterized in aerobically grown cells of Saccharomyces cerevisiae. The cells were grown under both high and low glucose concentrations, i.e., either in a chemostat at steady state with a specific growth rate of 0.1 h(-1) or in a batch culture with a specific growth rate of 0.37 h(-1). Experiments were carried out using [1-(13)C]glucose as the limiting substrate, and the resulting summed fractional labelings of intracellular metabolites were measured by gas chromatography coupled to mass spectrometry. The data were used as inputs to a flux estimation routine that involved appropriate mathematical modelling of the central carbon metabolism of S. cerevisiae. The results showed that the analysis is very robust, and it was possible to quantify the fluxes in the central carbon metabolism under both growth conditions. In the batch culture, 16.2 of every 100 molecules of glucose consumed by the cells entered the pentose-phosphate pathway, whereas the same relative flux was 44.2 per 100 molecules in the chemostat. The tricarboxylic acid cycle does not operate as a cycle in batch-growing cells, in contrast to the chemostat condition. Quantitative evidence was also found for threonine aldolase and malic enzyme activities, in accordance with published data. Disruption of the MIG1 gene did not cause changes in the metabolic network structure or in the flux pattern.

Acetyl Coenzyme A↗

PubMed2001

The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon.

The kidneys "escape" from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism.

Aldosterone↗