No effect of trans sodium crocetinate on maximal O(2) conductance or V(O(2),max) in moderate hypoxia

The lumped parameter describing skeletal muscle diffusional conductance for O(2), DM(O(2)), reflects all of the resistances for O(2) in moving from red cell to muscle fiber mitochondria. The purpose of our study was to determine if the carotenoid compound, trans sodium crocetinate (TSC), which has been reported to increase the diffusivity of O(2) in plasma, improves DM(O(2)) and thus, V(O(2),max) in maximally contracting in situ skeletal muscle. V(O(2),max) was measured in the isolated perfused canine gastrocnemius (n=5) during 3 min of isometric tetanic contractions at 1 Hz, while the animal was breathing 12% O(2) (PA(O(2))=32+/-2 Torr, mean+/-S.E.) under two experimental conditions. The first was a control contraction period and the second (following 60 min recovery) was performed within 5 min after infusion of a 0.1 mg x ml(-1) solution of TSC (total dose 100 microg kg(-1)). There were no significant differences in convective O(2) delivery (11.9+/-2.3 vs. 12.1+/-2.2 ml x min(-1) x 100 g(-1)), V(O(2),max) (9.5+/-1.5 vs. 9.6+/-1.5 ml x min(-1) x 100 g(-1)) or calculated DM(O(2)) (0.37+/-0.03 vs. 0.37+/-0.04 ml x min(-1) x 100 g(-1) x Torr(-1)) between contraction periods. As such, our results show that TSC does not improve performance in maximally contracting canine gastrocnemius muscle in situ under moderately hypoxic conditions, suggesting either that TSC in this situation does not increase plasma O(2) diffusivity or that this step in O(2) diffusion from red cell to myocyte does not constrain DM(O(2)).     

Oxygen diffusivity in tumor tissue (DS-Carcinosarcoma) under temperature conditions within the range of 20–40°C

Summary The O₂ diffusion constants D and K of tumor tissue (DS-Carcinosarcoma in the rat kidney) were determined at temperatures of 20, 30, 37, and 40°C. The following mean values were obtained for the conditions of 37°C: D=1.75·10^–5 cm²/s and K=1.9·10^–5 mlO₂/cm·min·atm. Within the range of 20–40°C, temperature variations in tumor tissue cause changes in the O₂ diffusion coefficient D of 2.0–2.5%/°C and in the Krogh O₂ diffusion constant K of 0.5–1.5%/°C. The measured O₂ diffusion constants for tumor tissue correspond to values of normal tissue with similar water content. This indicates that the insufficient O₂ supply in DS-Carcinosarcoma is due not to unfavorable O₂ diffusivity of the tumor tissue but rather to a decreased convective O₂ transport and to insufficient capillarization. An analysis of O₂ diffusion in DS-Carcinosarcoma tissue using the determined O₂ diffusion constants lead to the result that, under the conditions of arterial normoxia and normocapnia, critical O₂ supply conditions are to be expected when the intercapillary distance exceeds approximately 120 m.     

Diperoxovanadate can substitute for H(2)O(2) at much lower concentration in inducing features of premature cellular senescence in mouse fibroblasts (NIH3T3)

Stress induced premature senescence (SIPS) in mammalian cells is an accelerated ageing response and experimentally obtained on treatment of cells with high concentrations of H₂O₂, albeit at sub-lethal doses, because H₂O₂ gets depleted by abundant cellular catalase. In the present study diperoxovanadate (DPV) was used as it is known to be stable at physiological pH, to be catalase-resistant and to substitute for H₂O₂ in its activities at concentrations order of magnitudes lower. On treating NIH3T3 cells with DPV, SIPS-like morphology was observed along with an immediate response of rounding of the cells by disruption of actin cytoskeleton and transient G2/M arrest. DPV could bring about growth arrest and senescence associated features at 25 μM dose, which were not seen with similar doses of either H₂O₂ or vanadate. A minimal dose of 150 μM of H₂O₂ was required to induce similar affects as 25 μM DPV. Increase in senescent associated markers such as p21, HMGA2 and PAI-1 was more prominent in DPV treated cells compared to similar dose of H₂O₂. DPV-treated cells showed marked relocalization of Cyclin D1 from nucleus to cytoplasm. These results indicate that DPV, stable inorganic peroxide, is more efficient in inducing SIPS at lower concentrations compared to H₂O₂.     

Evaluation of p16INK4a, minichromosome maintenance protein 2, DNA topoisomerase IIalpha, ProEX C, and p16INK4a/ProEX C in cervical squamous intraepithelial lesions

p16INK4a has been shown to be overexpressed in nearly all high-grade squamous intraepithelial lesions (HSILs). Other cell-cycle regulators, such as minichromosome maintenance protein 2 (MCM2), DNA topoisomerase IIalpha (TOP IIA), and ProE(X) C (a cocktail of MCM2 and TOP IIA), have also demonstrated some value in identifying squamous intraepithelial lesions. Data on direct comparison of those cell regulatory proteins in the detection of squamous intraepithelial lesions, with a focus on low-grade squamous intraepithelial lesions (LSILs), are limited. We immunohistochemically evaluated the diagnostic value of p16, MCM2, TOP IIA, ProE(X) C, and a cocktail of p16 and ProE(X) C in 62 cervical biopsy specimens, including 14 cases of benign squamous mucosa (group 1), 34 cases of LSILs (group 2), and 14 cases of HSILs (group 3). The staining intensity and distribution were recorded. The results demonstrated that positive staining for p16 and the p16/ProE(X) C was observed in 100% of cases in group 3, whereas 79%, 86%, and 79% of cases were positive for CM2, TOP IIA, and ProE(X) C, respectively. ProE(X) C and the p16/ProE(X) C showed positive staining in 94% and 100% of cases in group 2, respectively. In contrast, immunoreactivity for p16, MCM2, and TOP IIA was detected in only 76% of cases in group 2. Importantly, all 8 p16-negative cases in group 2 were positive for p16/ProE(X) C (P = .003). Our data indicate that (1) p16 is a more sensitive and specific marker for identifying HSILs; (2) ProE(X) C is a better marker for the detection of LSILs; and (3) p16/ProE(X) C provides the highest diagnostic value for the detection of both HSILs and LSILs.     

Development of Fe3O4/Ag core/shell-based multifunctional immunomagnetic nanoparticles for isolation and detection of CD34+ stem cells

Fe₃O₄/Ag core/shell nanoparticles functionalized with the free amino (NH₂) functional groups (Fe₃O₄/Ag-NH₂) were conjugated with fluorescent electron coupled dye (ECD)-antiCD34 antibody using the 1-ethyl-3-(3′-dimethyl-aminopropyl) carbodiimide (EDC) catalyst (ECD – Electron Coupled Dye or R Phycoerythrin-Texas Red is a fluorescent organic dye attached to the antibody). The characteristic fluorescence of ECD in the antibody was investigated and was used as a good indicator for estimating the percentage of the antibodies that were successfully conjugated with the nanoparticles. The conjugation efficiency was found to increase depending on the V_NP:V_AB ratio, where V_NP and V_AB are the volumes of the nanoparticle solution (concentration of 50 ppm) and the as-purchased antibody solution, respectively. The conjugation efficiency rapidly increased from approximately 18% to approximately 70% when V_NP:V_AB was increased from 2:1 to 100:1, and it gradually reached the saturated state at an efficiency of 95%, as the V_NP:V_AB was equal to 300:1. The bioactivity of the abovementioned conjugation product (denoted by Fe₃O₄/Ag-antiCD34) was evaluated in an experiment for the collection of stem cells from bone marrow samples.     

Effect of a C/EBP gene replacement on mitochondrial biogenesis in fat cells

CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPbeta, are required for fat cell differentiation and maturation. Previous studies showed that replacement of C/EBPalpha with C/EBPbeta, generating the beta/beta alleles in the mouse genome, prevents lipid accumulation in white adipose tissue (WAT). In this study, beta/beta mice lived longer and had higher energy expenditure than their control littermates due to increased WAT energy oxidation. The WAT of beta/beta mice was enriched with metabolically active, thermogenic mitochondria known for energy burning. The beta/beta allele exerted its effect through the elevated expression of the G protein alpha stimulatory subunit (Galphas) in WAT. Galphas, when overexpressed in fat-laden 3T3-L1 cells, stimulated mitochondrial biogenesis similar to that seen in the WAT of beta/beta mice, and effectively diminished the stored lipid pool.     

Protein kinase C consensus sites and the regulation of renal Na/Pi-cotransport (NaPi-2) expressed in XENOPUS laevis oocytes

Renal brush border membrane sodium/phosphate (Na/P_i)-cotransport activity is inhibited by hormonal mechanisms involving activation of protein kinases A and C. The recently cloned rat renal Na/P_i cotransporter (NaPi-2) contains several protein kinase C but no protein kinase A consensus sites [17, 20]. In the present study we have expressed wild type and polymutant (protein kinase C consensus sites removed) NaPi-2-transporters in Xenopus laevis oocytes. The expression of transport function as well as the basic transport properties were unaffected by the removal of the consensus sites. Pharmacological activation of protein kinase C with phorbol 12,13-didecanoate (PDD) led to a time-dependent inhibition of expressed wild type Na/P_icotransport function; simultaneous exposure to staurosporine (0.3) prevented the PDD induced (50 nM) inhibition. The kinase-C-mediated inhibition was not prevented by the removal of the protein kinase C consensus sites. Pharmacological activation of protein kinase A (dibutyryl adenosine 3:5:cyclic monophosphate (cAMP)/forskolin) had no effect on wild type NaPi-2-induced oocyte Na/P_i cotransport. It is concluded that the protein-kinase-C-mediated regulation of expressed Na/P_i-cotransport does not involve the predicted consensus sites. The involvement of cryptic phosphorylation sites and/or of a phosphorylated regulatory protein is discussed.     

Oxygen transport in guinea pigs native to high altitude (Junin,Peru, 4,105 m)

In guinea pigs native to high altitude in the Andes (Peru) the arterial and mixed-venousP _{O 2},P _{CO 2}, pH, and O₂ content were measured at high altitude during breathing ambient air.Identical measurements were done in Nijmegen, The Netherlands, on sea-level natives and on guinea pigs exposed for 4–5 weeks to simulated altitude in a low pressure chamber, while breathing ambient air (normoxia) or an hypoxic mixture of O₂ in N₂ with aP _{I O 2} similar to that of the ambient air at high altitude. A standard blood O₂ dissociation curve (ODC) was estimated in vitro (at pH=7.4 and 37.5°C), and a standard in vivo ODC was derived from measuredP _{O 2},S _{O 2} and pH in all three groups.Both guinea pigs native to natural or simulated high altitude had a higher hematocrit and blood O₂ capacity than sea-level controls. These increased altitude values were, however, almost the same as the sea-level values of man or rat. No difference in the ideal alveolararterialP _{O 2} difference or lung diffusing capacity for O₂ was found between (natural or simulated) high altitude animals and their corresponding controls, when measured at hypoxia. Mixed-venousP _{O 2} was higher in guinea pigs from the natural high altitude (but not in those from the low pressure chamber) when compared with control sea-level natives studied at hypoxia. No difference among groups in cardiac output was found, while breathing the same inspiratory mixture. In the guinea pigs native to high altitude a higher P₅₀ and a lower Hill numbern for the in vitro ODC were found when compared with the controls or with the guinea pigs exposed to simulated high altitude. This was not observed when the ODC's were estimated in vivo.The rather modest polycythemic response to high altitude in guinea pigs coincides with a low value of P₅₀, when related to body weight. In this respect the guinea pig seems to be more closely related to the typical high altitude mammals like Andean camelids and rodents than to man or rat that respond to high altitude with a pronounced polycythemia and possess a rather high P₅₀ with respect to body weight.All data obtained in Peru are part of the scientific material acquired during the Italian Lake Mountain Scientific Expedition to Peru, march and April 1978, under the direction of Prof. Dr. P. G. Data from the University of Chieti, Italy.Part of the results were presented at the 12. Atmungsphysiologische Arbeitstagung, Göttingen, FRG, January 26–27, 1979     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.     

Direct evidence for GB virus C/hepatitis G virus (GBV-C/HGV) superinfection: elimination of resident viral strain by donor strain in a patient undergoing liver transplantation

The viral genome of GB virus C/hepatitis G virus (GBV-C/HGV), a single-strand RNA virus, is subject to considerable variability and at least four genotypes have been suggested based on phylogenetic analysis. While co-infection of GBV-C/HGV with other infectious agents such as hepatitis C virus (HCV) has been frequently observed, there is no report whether or not co-infection and/or superinfection occurs among different GBV-C/HGV strains. By studying a GBV-C/HGV positive recipient/donor pair in the context of undergoing liver transplantation, we have sequenced multiple clones derived from serum samples serially collected over four years. Detailed phylogenetic analyses have been performed with these sequences. The donor was infected with GBV-C/HGV genotype 1 and this strain completely replaced recipient GBV-C/HGV strain (genotype 2) after liver transplantation. The recipient's original viral strain became undetectable during follow-up. Sequence analysis failed to identify genetic recombination between the two genotypes, at least in whole structural domain. This study, therefore, provides direct evidence for GBV-C/HGV superinfection of one strain by another with one of them predominating probably due to replication competition.     

Preparation of hollow core/shell Fe3O4@graphene oxide composites as magnetic targeting drug nanocarriers

A main challenge for anticancer drugs is that the drugs can not arrive the cancer tissue at right time. In this work, a magnetic targeting nanoparticle based on hollow Fe₃O₄/graphene oxide (Fe₃O₄/GO) was developed as a potential tumor targeting drug carriers. The morphology results showed the Fe₃O₄ nanoparticles were uniformly wrapped by graphene oxide. After coating with graphene oxide, the Fe₃O₄/GO showed a higher saturation magnetization of 71.47 emu g^?1 as compared to neat Fe₃O₄ nanoparticles. The drug loading and releasing experiment indicated the obtained Fe₃O₄/GO has a good loading capacity of of 0.41 mg mg^?1 for 5-fluorouracil (5-FU) and a positive sensitive of acidic atmosphere. The CCK-8 assays of CMEC viability demonstrated the hollow Fe₃O₄/GO nanocarriers do not statistically exhibit toxicity with the concentration increasing from 2.5 to 40 μg mL^?1 in vitro. These results suggested the prepared Fe₃O₄/GO has a potential application in anticancer drugs nanocarriers.     

Cardiac output,arterial and mixed-venous O2 saturation,and blood O2 dissociation curve in growing rats adapted to a simulated altitude of 3500 m

Summary In rats adapted to a simulated altitude of 3500 m cardiac output measured at hypoxia by the direct Fick principle was significantly lower than in the control animals (mean values 54.3 ml/min and 69.8 ml/min, resp.). The decrease of cardiac output was accompanied by an increase of arterio-venous O₂ difference and a decrease of stroke volume in the adapted rats. It is suggested that the decrease of cardiac output might be related to the increase of hematocrit. The adapted rats also showed higher arterial and mixed-venous O₂ content (both at hypoxia) and increased O₂ capacity. Arterial O₂ saturation of the animals previously exposed to simulated high altitude hypoxia was significantly higher (67.3% as against 61.2% in the controls). The standard O₂ dissociation curve showed lower oxygen affinity in the blood of the adapted animals but no physiological advantage concerning the transport of O₂ to the tissues was found. In another group of animals the Bohr factor was estimated and no difference was found between rat and human blood.     

Coronary sinus outflow and O2 content in anterior cardiac vein blood at different levels of right ventricle performance

Summary Previous investigations have demonstrated that 60–70% of myocardial venous blood returns from the coronary sinus and that this percentage increases by experimentally raising right ventricle systolic pressure.The experiments described were performed in anesthetized open chest dogs and showed that the increase in coronary sinus outflow is not due to a change in the distribution of venous blood between the coronary sinus and the deep venous system, but is secondary to the increased left coronary artery inflow.A possible explanation of this might be that the increase in intramyocardial right ventricular pressure brings about some release of certain catecholamines which may induce a persisting increase in coronary artery inflow. Beta-blockade can in effect prevent any change in coronary blood flow arising from overdistension of the right ventricle.The O₂ content of the coronary sinus blood (returning mainly from the left ventricle) is constantly lower than that of the anterior cardiac and Thebesius veins (flowing mainly out of the right ventricle). This difference is very probably the result of mechanical factors which play an active role in regulating coronary flow. As is well known, total coronary resistances are the sum of two components i.e., a) extravascular compression during ventricular systole and b) active vasomotor tone in the coronary bed.Vasomotility in the right and left coronary arteries is virtually identical, whereas myocardial compression is considerably lower in the right side of the heart than in the left. Right coronary flow is therefore less impaired than the flow in the left coronary artery; the ratio between coronary flow and O₂ myocardial extraction should thus be better in the right than in the left ventricle.     

Phagocytic signaling strategies: Fcγreceptor-mediated phagocytosis as a model system

Phagocytosis is a phylogenetically ancient process by which eukaryotic cells engulf insoluble substances whose size exceeds approximately 0.5 μ m. The engulfment process requires the concerted action of several fundamental cellular pathways and is governed by multiple transmembrane signaling events. Here we focus on phagocytosis mediated by a well-studied class of phagocytic receptors that recognize the Fc portion of IgG (Fc_γRs ).     

(−)-Epigallocatechin-3-O-gallate (EGCG) reverses caffeine-induced anxiogenic-like effects

This study was designed to determine whether (−)-epigallocatethin-3-O-gallate (EGCG) could reverse caffeine-induced anxiogenic-like effects in animals. In mice, EGCG antagonized the caffeine-induced reduction in both the open arm entry number and time-spent in open arm on elevated plus-maze. In addition, EGCG also antagonized the caffeine-induced reduction in both the central zone distance and central zone time-spent on an open field apparatus, respectively. Electroencephalogram (EEG) was recorded from the rat anterior cerebral cortex. Caffeine increased the power density-ratios of fast (FW: 8.00–20.00 Hz) and slow (SW: 0.75–8.00 Hz) frequency spectrum bands in these EEG recordings. However, EGCG reduced the caffeine-induced increase of FW/SW ratios. Thus, EGCG reverses caffeine-induced anxiogenic-like effects. We also provide additional evidence that the EEG FW/SW (or SW/FW) ratios can be a useful tool for the prediction of anxiogenic and/or anxiolytic effects in an animal model.     

Hunger and satiety in genetically obese mice (C57BL/6J-ob/ob)

To determine the mechanism for hyperphagia in genetically obese mice (C57BL/6J-ob/ob), several experiments were conducted on the ability of these mice to respond to caloric deficits and surpluses. Presentation of food or sugar reduces subsequent operant licking in both obese and lean mice. When given sugar solutions, evaporated milk, or sweetened non-fat milk, both obese and lean mice reduce food intake to compensate for the calories obtained from the solutions. These findings indicate that genetically obese mice respond normally to caloric surpluses. Obese mice respond to food deprivation (caloric deficit) by increasing subsequent food intake but they do so more slowly than controls.     

Energy/speed relation of below-knee amputees walking on a motor-driven treadmill

Little is known about the energy/speed relation of those who are handicapped in the lower-limbs. In fact, this is true for users of below-knee prostheses. The importance of the above relation lies in the pronouncement that can be made in the matter of the results of a rehabilitation program, on the ground of the O₂-consumption during walking.In order to determine the relation between O₂-consumption and the walking speed, we had at our disposal 54 users of below-knee prostheses, all but a few disabled in consequence of a trauma.Thirty-seven subjects could reach and maintain walking speeds of 50 up to 90 m/min for a prolonged period.For this range of speeds equations for Gross and Nett /kg were determined. The measure of physical fitness as well as the cardiorespiratory functions and the conditions of stump and prosthesis were evaluated.The conclusion is that otherwise healthy users of below-knee prostheses are consuming approximately 20% more oxygen at all speeds than normal subjects. Stress is laid on the importance of these findings with respect to the rehabilitation. Furthermore, attention is invited for the necessity of maintaining or improving the physical condition apart from the specific walking training for these handicapped.     

Role of O2 in Regulation of Lactate Dynamics during Hypoxia: Mathematical Model and Analysis

The mechanistic basis of the relationship between O₂ and lactate concentration in muscle is not fully understood. Although hypoxia can cause lactate (LA) accumulation, it is possible for LA accumulation to occur without hypoxia. Nevertheless, during conditions of low O₂ availability, blood and tissue LA accumulation are used as indicators of hypoxia. To provide a framework for analyzing changes in energy metabolism and its regulation, we developed a mathematical model of human bioenergetics that links cellular metabolic processes to whole-body responses. Our model is based on dynamic mass balances and mechanistic kinetics in muscle, splanchnic and other body tissues for many substrates (glycogen, glucose, pyruvate, LA, O₂, CO₂, etc.) and control metabolites (e.g., ATP) through coupled reaction processes. Normal substrate concentrations in blood and tissues as well as model parameters are obtained directly or estimated indirectly from physiological observation in the literature. The model equations are solved numerically to simulate substrate concentration changes in tissues in response to disturbances. One key objective is to examine and quantify the mechanisms that control LA accumulation when O₂ availability to the muscle is lowered. Another objective is to quantify the contribution of different tissues to an observed increase in blood lactate concentration. Simulations of system responses to respiratory hypoxia were examined and compared to physiological observations. Model simulations show patterns of change for substrates and control metabolites that behave similarly to those found experimentally. From the simulations, it is evident that a large decrease can occur in muscle O₂ concentration, without affecting muscle respiration ( ) significantly. However, a small decrease in (1%–2%) can result in a large increase in LA production (50%–100%). The cellular rate of oxygen consumption, , which is coupled to ATP formation and NADH oxidation, can regulate other processes (e.g., glycolysis, pyruvate reduction) with high sensitivity through its effects on ADP/ATP and NADH/NAD. Thus, although LA metabolism does not depend directly on O₂ concentration, it is indirectly affected by , through changes in ADP/ATP, and NADH/NAD. Arterial LA concentration (C_a,LA) follows the pattern of change of muscle LA concentration (C_m,LA). Nevertheless, changes in C_a,LA, due to C_m,LA, are unlikely to be detected experimentally because changes in C_m,LA are small relative to the total LA concentrations in other tissues. © 1998 Biomedical Engineering Society. PAC98: 8710+e, 8722Fy