Effects of mitogens on sodium-potassium transport, H-ouabain binding, and adenosine triphosphatase activity in lymphocytes

The effect of various mitogens was studied on sodium (Na⁺) potassium (K⁺) transport, ³H-ouabain binding, and adenosine triphosphatase (ATPase) activity in human and sheep peripheral lymphocytes. Concanavalin A (ConA), phytohemagglutinin (PHA), horse anti-lymphocyte serum (ALS), and anti-IgG antisera, in order of decreasing potency, stimulated in particular the ouabain-sensitive K⁺ pump influx, while the cardiac glycoside-insensitive K⁺ leak flux was only slightly affected. Sheep lymphocytes primed in vivo with human IgG as antigen also responded with K⁺ pump flux activation when exposed to the antigen in vitro. Both PHA and ConA also stimulated active Na⁺ efflux in human lymphocytes. Apparently these mitogens activate the Na⁺K⁺ pump system in the lymphocyte membrane—an assumption supported by the finding of a significant activation of the ouabain-sensitive Na⁺K⁺-ATPase. From rate studies of ³H-ouabain binding carried out at 37 °C in presence and absence of sodium azide, and at 0 °C, it is concluded that PHA alters the rate of ouabain uptake to these cells. Thus PHA may alter the affinity of the pump for ouabain, equivalent to an increased cation turnover per pump site. However, our findings do not completely discount the possibility that PHA also increases the total number of ouabain molecules bound and therefore of Na⁺K⁺ pumps.     

Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep

Reticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence-staining techniques 5-6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M-cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain-sensitive K+ pump and ouabain-insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll-density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.     

Alkaline pH and Internal Calcium Increase Na+ and K+ Effluxes in LK Sheep Red Blood Cells in Cl−-free Solutions

We examined the effects of pH, internal ionized Ca (Ca²⁺ _i ), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K⁺ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K⁺ phenotypes. In LK SRBCs the K⁺ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K⁺ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K⁺ efflux was activated (100%) when Ca²⁺ _i was increased (+A23187, +Ca²⁺ _o ) into the μm range. However, in comparison to human red blood cells, the Ca²⁺ _i -induced increase in K⁺ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na⁺ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca²⁺ _i . In contrast, in HK SRBCs the K⁺ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca²⁺ _i . These studies suggest the presence in LK SRBCs, of at least two pathways for Cl⁻-independent K⁺ and Na⁺ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca²⁺ _i . Received: 23 May 1996/Revised: 6 December 1996     

Microbial preparation of metal-substituted magnetite nanoparticles

A microbial process that exploits the ability of iron-reducing microorganisms to produce copious amounts of extra-cellular metal (M)-substituted magnetite nanoparticles using akaganeite and dopants of dissolved form has previously been reported. The objectives of this study were to develop methods for producing M-substituted magnetite nanoparticles with a high rate of metal substitution by biological processes and to identify factors affecting the production of nano-crystals. The thermophilic and psychrotolerant iron-reducing bacteria had the ability to form M-substituted magnetite nano-crystals (M(y)Fe(3-y)O(4)) from a doped precursor, mixed-M iron oxyhydroxide, (M(x)Fe(1-x)OOH, x< or =0.5, M is Mn, Zn, Ni, Co and Cr). Within the range of 0.01< or =x< or =0.3, using the mixed precursor material enabled the microbial synthesis of more heavily substituted magnetite compared to the previous method, in which the precursor was pure akaganeite and the dopants were present as soluble metal salts. The mixed precursor method was especially advantageous in the case of toxic metals such as Cr and Ni. Also this new method increased the production rate and magnetic properties of the product, while improving crystallinity, size control and scalability.     

Genetic structure of the beaked whale genus Berardius in the North Pacific,with genetic evidence for a new species

There are two recognized species in the genus Berardius, Baird's and Arnoux's beaked whales. In Japan, whalers have traditionally recognized two forms of Baird's beaked whales, the common “slate‐gray” form and a smaller, rare “black” form. Previous comparison of mtDNA control region sequences from three black specimens to gray specimens around Japan indicated that the two forms comprise different stocks and potentially different species. We have expanded sampling to include control region haplotypes of 178 Baird's beaked whales from across their range in the North Pacific. We identified five additional specimens of the black form from the Aleutian Islands and Bering Sea, for a total of eight “black” specimens. The divergence between mtDNA haplotypes of the black and gray forms of Baird's beaked whale was greater than their divergence from the congeneric Arnoux's beaked whale found in the Southern Ocean, and similar to that observed among other congeneric beaked whale species. Taken together, genetic evidence from specimens in Japan and across the North Pacific, combined with evidence of smaller adult body size, indicate presence of an unnamed species of Berardius in the North Pacific.     

Large-scale production of magnetic nanoparticles using bacterial fermentation

Production of both nano-sized particles of crystalline pure phase magnetite and magnetite substituted with Co, Ni, Cr, Mn, Zn or the rare earths for some of the Fe has been demonstrated using microbial processes. This microbial production of magnetic nanoparticles can be achieved in large quantities and at low cost. In these experiments, over 1 kg (wet weight) of Zn-substituted magnetite (nominal composition of Zn_0.6Fe_2.4O₄) was recovered from 30 l fermentations. Transmission electron microscopy (TEM) was used to confirm that the extracellular magnetites exhibited good mono-dispersity. TEM results also showed a highly reproducible particle size and corroborated average crystallite size (ACS) of 13.1 ± 0.8 nm determined through X-ray diffraction (N = 7) at a 99% confidence level. Based on scale-up experiments performed using a 35-l reactor, the increase in ACS reproducibility may be attributed to a combination of factors including an increase of electron donor input, availability of divalent substitution metal ions and fewer ferrous ions in the case of substituted magnetite, and increased reactor volume overcoming differences in each batch. Commercial nanometer sized magnetite (25–50 nm) may cost $500/kg. However, microbial processes are potentially capable of producing 5–90 nm pure or substituted magnetites at a fraction of the cost of traditional chemical synthesis. While there are numerous approaches for the synthesis of nanoparticles, bacterial fermentation of magnetite or metal-substituted magnetite may represent an advantageous manufacturing technology with respect to yield, reproducibility and scalable synthesis with low costs at low energy input.     

Ouabain-insensitive sodium/lithium exchange and the effect of anti-L in low potassium sheep erythrocytes

The activity of the ouabain-insensitive Na⁺/Na⁺ exchange system was assessed by measurements of Li⁺ net-uptake in LK and HK sheep erythrocytes in the absence and presence of the L-antibody and various inhibitors. N-ethylmaleimide, p-chloromercuribenzoic sulfonate and phloretin inhibited the exchange by about 50%. Anti-L, while stimulating the K⁺ pump flux in LK cells, did not alter Na⁺/Li⁺ countertransport. The activity of the exchange system with fully saturated internal and external loading sites was estimated to be identical in LK and HK sheep red cells. Hence the Na⁺/Na⁺ exchange system seems to be molecularly unrelated to the ouabain-sensitive Na⁺K⁺ pump in these cells and not under genetic control of the HK/LK and M/L genes.     

Malaria vectors and transmission dynamics in coastal south-western Cameroon

Background

Malaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas.

Methods

A 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months.

Results

In all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species.

Conclusion

Malaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa.

     

Pig reticulocytes. V. Development of Rb+ influx during in vitro maturation

Influx of the K⁺ analogue Rb⁺ was measured through the ouabain-sensitive Na⁺/K⁺ pump and the ouabain-insensitive “leak” pathways in Cl^? or NO in mature red cells from adult pigs and in reticulocytes naturally occurring in 7-day-old piglets. In reticulocytes, Rb⁺ influxes by the two pathways were of about equal magnitude in Cl^? (13 and 10 mmoles/liter cells × hr) and at least 25-fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na ⁺ media, a portion of the ouabain-insensitive “leak” flux of Rb⁺ was Cl^? dependent (Rb⁺Cl^? transport) as NO replacement reduced Rb⁺ influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N-ethylmaleimide (NEM) stimulated Rb⁺Cl^? transport about twofold in reticulocytes and up to 13-fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain-sensitive Rb⁺ pump and ouabain-insensitive Rb⁺Cl^? influx were lost. In contrast, the NEM-stimulated Rb⁺Cl^? transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb⁺Cl^? transport. Although the experimental variability precluded a full assessment of significant changes in the small Na⁺/K⁺(Rb⁺) pump and Rb⁺Cl^? fluxes in mature pig red cells kept for the same time period in vitro, Rb⁺ flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity transitions of Na⁺/K⁺ pump and Cl^?-dependent K⁺ fluxes in vivo.