Furosemide-sensitive Na+-K+ cotransport and cellular metabolism in human erythrocytes

Metabolic depletion of human red cells with 2-deoxy-D-glucose in the presence of EGTA decreased ATP to about 4% of the initial value and increased total ouabain- and furosemide-resistant Na+ and K+ effluxes by 20% and 100%, respectively, and furosemide-sensitive Na+ and K+ effluxes by 100% and 60%, respectively. When ATP was restored, all the components of Na+ and K+ fluxes measured returned to baseline levels suggesting a metabolic dependence.     

K:Cl cotransport: emerging molecular aspects of a ouabain-resistant, volume-responsive transport system in red blood cells

Erythrocytes from teleosts to mammals, like man, possess a ouabain-resistant K flux component which is Cl-dependent and activated upon suspension of the cells into hyposmotic media. This volume-responsive K:Cl cotransporter may be synonymous with a Cl-dependent K pathway stimulated by chemical interventions such as SH group (thiol) alkylation or oxidation at cytoplasmic sites of the membrane. Based on a comparison of the response of the two activity expressions of probably the same molecule to ionic and metabolic perturbations a molecular model is proposed accommodating K:Cl flux activities under a variety of stimulatory and inhibitory influences. Physiologically, this pathway may be involved in the maturational cell volume reduction occurring between the stages of reticulocytes and the final erythrocytes as well as corresponsible for the expression of the low K steady state of certain animal red blood cells. The pathophysiological potential of this K:Cl transporter lies in the fact that, when activated, red blood cells dehydrate through K:Cl loss.     

Stimulation of active potassium transport in LK sheep red cells by blood group-L-antiserum

Summary Anti-L serum prepared by immunization of a high-potassium-type (HK) (blood type MM) sheep with blood from a low-potassium-type (LK) (blood type ML) sheep contained an antibody which stimulated four- to sixfold K⁺-pump influx in LK (LL) sheep red cells. In long-termin vitro incubation experiments, LK sheep red cells sensitized with anti-L showed a net increase in K⁺ after two days of incubation at 37°C, whereas HK-nonimmune (NI)-serum-treated control cells lost K⁺. The antibody could be absorbed by LK (LL) sheep red cells but not by HK sheep red cells. Kinetic experiments showed that the concentration of external K⁺ ([K⁺]₀) required to produce halfmaximum stimulation of the pump ([Na⁺]₀=0, replaced by Mg⁺⁺) was the same (0.25 mM) in L-antiserum-treated or untreated LK cells. LK cells with different [K⁺]_i (Na⁺ replacement) were prepared by the p-chloromercuribenzene sulfonate (PCMBS) method. At [K⁺]₀=5 mM, pump influx decreased as [K⁺]_i increased from 1 to 70 mM in L-antiserum-treated LK cells, whereas LK cells treated with HK-NI-serum ceased to pump at [K⁺]_i=35 mM. Exposure to anti-L serum produced an almost twofold increase in the number of pump sites of LK cells as measured by the binding of tritiated ouabain by LK sheep red cells. These findings indicate that the formation of a complex between the L-antigen and its antibody stimulates active transport in LK sheep red cells both by changing the kinetics of the pump and by increasing the number of pump sites.     

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%± 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%± 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.     

The M-antigen in HK and LK sheep red cell membranes

Summary Red cells of all high-potassium-type (HK) sheep and of more than one half of all low-potassium-type (LK) sheep contained the M-antigen and were hemolyzed by iso-immune anti-M antiserum in presence of a guinea pig serum complement. It was characteristic for the hemolysis of HK red cells by the M-antiserum the all HK cells were ultimately hemolyzed at suboptimal antibody concentrations, provided the time of incubation at 37 °C was sufficiently long. Thus, the M-antigen appears to be expressed on all red cells of an individual HK sheep. The M-antibody was absorbed by HK red cells and their membranes with a high affinity, whereas M-negative LK red cells and their membranes did not bind the antibody. The ratio of the number of antibody units absorbed per cell or membrane to the number of antibody units required for lysis approached unity. The amount of antibody absorbed per membrane was unaffected by ouabain in the presence of ATP, Mg⁺⁺, Na⁺, and K⁺. The M-antigen activity depends on the integrity of the red cell membrane and was not detectable after lyophilization of HK membranes or in the membrane protein solubilized by n-butanol. The major M-antibody activity was found among the high molecular weight plasma proteins and may be attributed to the ₂ M globulins. Heterogeneity within the antibody fraction cannot be excluded since some hemolytic activity was detected in a chromatographic fraction containing predominantly -globulin. The relationship between the M-antigen and the Na⁺–K⁺ transport system in sheep red cell membranes is discussed.This work was presented in part at the 53rd annual meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N. J. 1969.     

Impacts of cage culture on physico-chemical and bacteriological water quality in Lake Volta,Ghana

The effects of cage fish farming on physico-chemical and bacteriological water quality in Lake Volta, Ghana, were investigated in 2013–2014. Farmed and unfarmed (control) areas of the lake were selected for monitoring. Nutrients, temperature, dissolved oxygen, conductivity, turbidity, pH, total coliforms, Pseudomonas and Vibrio spp. in the water were monitored monthly. Analyses of the water samples were carried out according to standard procedures. Physico-chemical quality of the water in both farm and control sites were within ranges typical of minimally impacted water and did not vary significantly between the two contrasting sites. The bacteriological analysis, however, revealed contamination of the lake water by fish farming. The bacterial counts at the farmed sites were significantly higher (p < 0.05) than those of the control sites, with figures at the farmed sites ranging from 132 to 1 708 cfu 100 ml?1 for total coliforms, 514 to 5 170 cfu 100 ml?1 Pseudomonas spp. and 14 to 516 cfu 100 ml?1 for Vibrio spp. The results suggested that cage fish farming has increased bacterial loads in the lake water, but has had minimal impact on its physico-chemical quality.