Studies on the electron transport chain at subzero temperatures: electron transport at site 3.
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The presence of the ammonia-oxidising group Nitrosomonas in developing biofilms from a subsurface flow dairy effluent treatment wetland was investigated using in situ hybridisation and a crude semi-quantitative PCR. Fluorescently labelled oligonucleotide probes, specific for the Nitrosomonas europaea and Nitrosomonas eutropha species, showed that nitrifying bacteria were present from the onset of biofilm development but that over a five month period the proportions remained low (averaging less than 1% of the total bacterial population). The greatest presence of these nitrifiers was found in the second trench of the subsurface flow wetland. Nitrosomonas were also found to be susceptible to environmental change; adverse environmental conditions, such as low temperatures, coincided with negligible Nitrosomonas presence throughout the wetland. However, during one period of elevated ammonium concentrations Nitrosomonas presence rapidly increased as high as 7% of the total bacterial population. Investigations of relative occurrence of effluent bacterial population of Nitrosomonas europaea and the biofilm populations showed that Nitrosomonas presence in the effluent coincided with significant presence in adjacent biofilms. This suggests seeding between both sessile and planktonic populations of this species. Nitrosomonas occurred as single cells in biofilms throughout the study and were never observed as dense clusters.
Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electrophoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and lambda 1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.
OBJECTIVE: To investigate the expression of endothelin (ET)-1 and its receptors in the cerebral arterioles of stroke-prone (spSHR) and control spontaneously hypertensive rats (SHRs) and the changes in endothelin receptor subtypes A and B density elicited by a stroke-permissive diet, before the development of stroke. METHODS: Six-week-old SHRs (n=11) and spSHRs (n=11) were assigned to either a regular or a "Japanese"-style diet, in addition to 1% NaCl in the drinking water, for 4 weeks. Cryosections (10 microm thick) of rat brain were assessed for endothelin receptor distribution and density by autoradiography with [125I]ET-1 (10(-10) mol/l) in the presence of cold ET-1 (10(-6) mol/l) or the peptide antagonists BQ-123 (10(-6) mol/l) or BQ-788 (10(-6) mol/l). Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect specific mRNAs and localize immunoreactive ET-1 and ET(A) and ET(B). RESULTS: In both strains, immunoreactive ET-1 was detected in the endothelium of cerebral arterioles, and RT-PCR and autoradiography demonstrated the coexistence of both receptor subtypes in brain homogenates and the cerebral arteriole walls, respectively. With the regular diet, the ET(A) receptor density was lower in SHRs than in spSHRs (P = 0.007), whereas the ET(B) receptor density was similar (P = NS). The Japanese-style diet increased the density of ET(A) receptors (P = 0.006) in SHRs, but decreased it (P = 0.019) in spSHRs. No effect was seen on ET(B) receptor density. CONCLUSIONS: ET(A) and ET(B) receptor subtypes are expressed in the wall of cerebral arterioles of SHRs and spSHRs. The latter strain showed a marked increase in ET(A) receptor density under a regular diet, in addition to an altered regulation in response to a stroke-permissive diet.
The geographic mosaic theory of coevolution posits that the form of selection between interacting species varies across a landscape with coevolution important and active in some locations (i.e., coevolutionary hotspots) but not in others (i.e., coevolutionary coldspots). We tested the hypothesis that the presence of red squirrels (Tamiasciurus hudsonicus) affects the occurrence of coevolution between red crossbills (Loxia curvirostra complex) and Rocky Mountain lodgepole pine (Pinus contorta ssp. latifolia) and thereby provides a mechanism giving rise to a geographic mosaic of selection. Red squirrels are the predominant predispersal seed predator and selective agent on lodgepole pine cones. However, in four isolated mountain ranges east and west of the Rocky Mountains, red squirrels are absent and red crossbills are the main predispersal seed predator. These isolated populations of pine have apparently evolved without Tamiasciurus for about 10,000 to 12,000 years. Based on published morphological, genetic, and paleobotanical studies, we infer that cone traits in these isolated populations that show parallel differences from cones in the Rocky Mountains have changed in parallel. We used data on crossbill and conifer cone morphology and feeding preferences and efficiency to detect whether red crossbills and lodgepole pine exhibit reciprocal adaptations, which would imply coevolution. Cone traits that act to deter Tamiasciurus and result in high ratios of cone mass to seed mass were less developed in the isolated populations. Cone traits that act to deter crossbills include larger and thicker scales and perhaps increased overlap between successive scales and were enhanced in the isolated populations. In the larger, isolated mountain ranges crossbills have evolved deeper, shorter, and therefore more decurved bills to exploit these cones. This provides crossbills with higher feeding rates, and the change in bill shape has improved efficiency by reducing the concomitant increases in body mass and daily energy expenditures that would have resulted if only bill size had increased. These parallel adaptations and counter adaptations in red crossbills and lodgepole pine are interpreted as reciprocal adaptations and imply that these crossbills and pine are in coevolutionary arms races where red squirrels are absent (i.e., coevolutionary hotspots) but not where red squirrels are present (i.e., coevolutionary cold-spots).
Several lines of evidence suggest that central serotoninergic neurons may mediate fatigue signals during prolonged exercise. In this study we examined the effects of diet and ambient temperature on peripheral modulators and indices of serotoninergic function and their relationship to exercise performance. Six well-trained cyclists participated, in randomised order, in two diet and exercise regimens each lasting 8 days and comprising four cycle tests to exhaustion at 70 % of maximum oxygen uptake. On days 1 and 5, subjects exercised to exhaustion to deplete muscle glycogen. For 3 days after the first depletion trial a diet providing 10 % of energy in the form of carbohydrate (CHO) was consumed (low CHO), and for 3 days after the second depletion trial a diet providing 80 % (high CHO) of energy as CHO was consumed, and each diet was followed by a performance trial at the same ambient temperature, either 10 degrees C or 30 degrees C (days 4 and 8). This schedule was repeated after 1 week, but performance trials were carried out at the other ambient temperature. In the cold, cycling time increased (P < 0.01) from 89.2 (78.0-129.5) min (median (range)) in the low CHO trial to 158.2 (116.9-165.6) min in the high CHO trial. In the heat, cycling time increased from 44.0 (31.8-51.4) min in the low CHO trial to 53.2 (50.2-82.2) min on the high CHO trial (P = 0.02). The serum prolactin (Prl) concentration was higher at exhaustion during the two trials in the heat than in the two trials in the cold. Serum Prl levels were unrelated to the purported peripheral modulators of serotoninergic function (plasma concentrations of total tryptophan (Trp), free Trp, branched-chain amino acids (BCAAs), free Trp/BCAA ratio and total Trp/BCAA ratio) but were significantly related to the rectal temperatures measured during the two trials in the heat. This finding provides indirect evidence that the serotoninergic system may be involved in fatigue during exercise under conditions of heat stress.
Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).
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Pathological clones of plasma cells produce monoclonal immunoglobulins (M-Ig), either the entire molecule or only light chains. These proteins lead in the organism to various pathological process. The increased blood viscosity causes eyesight damage. Impaired coagulation or haemolysis caused by cold agglutinins are frequent. Depending on the orientation of the antigenic determinant of M-Ig, nervous and muscular structures suffer damage, as well as the kidney; metabolic disorders, hypoglycaemia or endocrinopathies may develop. Some of the mentioned disorders are readily diagnosed, some can be detected only by an aimed examination. The presented review should help early diagnosis of these pathological processes.
OBJECTIVE: To determine the gene sequence and tissue distribution of uncoupling protein 3 (Ucp3) in the collared lemming, we quantified mRNA expression of Ucp3 under known states of altered energy expenditure (photoperiod-induced weight gain and loss, cold exposure, and fasting) and measured mitochondrial oxygen consumption to assess possible functional changes in energy expenditure. RESEARCH METHODS AND PROCEDURES: The Ucp3 gene sequence information was obtained using the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. Northern blots were used to determine mRNA expression levels. Respirometry was used to measure oxygen consumption rates in isolated mitochondria. RESULTS: The lemming Ucp3 gene has a 97% sequence similarity with other published Ucp3 sequences at the amino acid level. Ucp3 mRNA is expressed in muscle, heart, and brown adipose tissue of collared lemmings. Long-photoperiod lemmings have a higher expression of Ucp3 mRNA than short-photoperiod lemmings (p < 0.001) in both muscle and brown adipose tissue. Transferring lemmings from long to short photoperiods (inducing weight gain) significantly decreased Ucp3 mRNA expression (p < 0.01), whereas transferring lemmings from short to long photoperiods (inducing weight loss) significantly increased Ucp3 expression (p < 0.001). Muscle Ucp3 mRNA expression was significantly decreased by 10 days of mild (10 degrees C) cold exposure (p < 0.001). Muscle Ucp3 mRNA expression was significantly increased by fasting (p < 0.01) and was correlated to plasma free fatty acid levels (r = 0.7). Photoperiod transfer did not alter mitochondrial coupling. DISCUSSION: These data suggest that UCP3 may not be involved in energy expenditure in the collared lemming.
Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP). Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing. DNA-RFLP resolved 19 of the 22 meat strains into 11 groups. Three meat strains were untypable using this method. All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes. With PCR-RFLP, the 22 meat strains produced eight distinct genotypes. 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence. PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used. Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species. Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms. The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.
Isolated brown adipocytes were prepared from guinea pigs acclimated to 28 degrees C or exposed to 4-8 degrees C for periods of up to 3 wk. Cells from warm-adapted animals retained respiratory control when stimulated with norepinephrine. Cells from guinea pigs exposed to cold for 4-21 days showed a much greater respiratory response to norepinephrine due to enhanced uncoupling rather than enhanced substrate supply. After 7 days of cold acclimation, norepinephrine-stimulated respiration became uncontrolled and was limited only by the maximal respiratory capacity of the mitochondria. Three weeks of cold acclimation were accompanied by a doubling of total cell number, a doubling of the mitochondrial protein per adipocyte, and a sixfold increase in the norepinephrine-stimulated respiration per in situ mitochondrion with no change in respiratory chain capacity. The induction of norepinephrine-stimulated respiration correlated with the appearance of high-affinity purine nucleotide binding sites on the mitochondria, diagnostic of the uncoupling protein. If the results are extrapolated to the whole animal, they indicate that brown adipose tissue makes little contribution to thermogenesis in the warm-adapted guinea pig but may account for most or all the increment seen on cold adaptation.
Muscle is a very adaptable tissue in which gene expression is to a large extent influenced by physical signals. Adaptation to a different work regime is brought about by changes in fibre type and fibre cross-sectional area. We have shown both mass and phenotype are markedly altered by stretch and force production within a period as short as 4 days. This is associated with quantitative as well as qualitative changes in gene expression. The latter involves the expression of myosin heavy chain isogenes which encode different types of molecular motors. Some species of fish have exploited this and they are able to rebuild their myofibrillar systems for warm and cold temperature swimming by selective myosin gene expression. To understand how the different myosin isoform confer different contractile properties methods have been developed for cloning, sequencing and visualizing the structure of the ATPase site to explain how the molecular motors are designed. With regard to the chemical link between the physical signal and the upregulation of certain muscle genes we have cloned a new growth factor that is only expressed in muscles subjected to stretch and/or exercise and which is designed for autocrine/paracrine action. Experiments indicate that the expression of a local growth factor which induces repair, remodelling and hypertrophy is one of the ways cells respond to mechanical strain.
INTRODUCTION: Inflammatory events within the gut muscularis contribute to dysmotility. We hypothesized that manipulation during organ harvesting initiated an inflammatory response via muscularis macrophages and that this cascade was amplified during reperfusion. METHODS: Small bowel transplantation was performed in Lewis rats. To investigate the impact of organ harvesting on muscularis inflammation, cold whole-body perfusion was performed after versus prior to organ harvesting. The role of macrophages was investigated by transplantation of the macrophage-depleted gut. Leukocyte infiltration was investigated in muscularis whole mounts. Mediator mRNA expression was determined by real-time reverse transcriptase polymerase chain reaction. Contractility was assessed in a standard organ bath. RESULTS: Organ harvesting and ischemia-reperfusion induced leukocyte recruitment and mRNA upregulation in the muscularis: interleukin-6 12217-fold, monocyte chemoattractant protein-1 62-fold, ICAM-1 12-fold, cyclooxygenase-2: 8-fold, iNOS: 150-fold. Although organ harvesting with cold ischemia prevented early gene expression, peak expression at 3-hour reperfusion was not changed by modification of the harvesting technique. Compared to controls, transplanted animals showed a 63% decrease in smooth muscle contractility. In contrast, transplanted macrophage-depleted gut exhibited significantly fewer leukocytes and only a 16% decrease in contractility. CONCLUSIONS: Gut manipulation during organ harvesting initiates an inflammatory response within the muscularis that is massively intensified during reperfusion. This change contributes to muscular dysfunction. Furthermore, the results suggested that resident macrophages play a key role in initiating this process.