The effect of ferricyanide with iodoacetate in calcium-free solution on passive cation permeability in human red blood cells: comparison with the Gardos-effect and with the influence of PCMBS on passive cation permeability

Freshly prepared human red blood cells incubated with 5 mM ferricyanide, 0.2 mM iodoacetate and 2 mM adenosine in the presence of 5 mM EGTA demonstrate comparable increases in Na+ and K+ permeability (ferricyanide effect). This effect is unrelated to the Ca2+-activated K+ channel (Gardos effect) since influx of Ca2+ from outside the cell is excluded. Also this effect is different from the non-specific Na+ and K+ permeability change elicited by PCMBS. These differences become obvious by using various reagents. For example, A23187 and quinidine exert opposite effects in Gardos and ferricyanide experiments, where A23187 and atebrin react oppositely in the latter and in PCMBS experiments. The ferricyanide effect described here does not involve formation of nonspecific channels. The change in Na+ permeability separately from K+ permeability under certain circumstances suggests a more specific effect.     

Expression of interleukin 2 and the interleukin 2 receptor in aging rats

Lymphocytes of aged animals exhibit a marked decrease in proliferative capacity in response to mitogen stimulation when compared to those of younger animals. In humans and mice the decreased proliferation is due at least in part (i) to the inability of lymphocytes to synthesize sufficient interleukin 2 (IL-2) and (ii) to decreased expression of IL-2 receptors (IL-2R) on the surface of aged lymphocytes. We compared proliferative abilities, IL-2 production, and IL-2R expression in splenocyte cultures of 4- to 5- and 22- to 24-month-old Fischer 344 rats stimulated with either concanavalin A (Con A) or A23187 and phorbol myristate acetate (PMA). Proliferation was significantly decreased in aged lymphocytes (30-50%) with both treatment protocols. However, unlike mice and humans we observed no difference in IL-2 activity, IL-2 mRNA levels, or IL-2R cell surface expression of lymphocytes from young and aged rats stimulated with either Con A or A23187 and PMA. These results indicate that factors other than decreased expression of IL-2 and IL-2R are responsible for the diminished proliferative capacity of aged rat lymphocytes following mitogen stimulation.     

Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.     

The superficial cerebral veins

Estimated were the number, the course, and the width of the superficial cerebral veins. The veins on the superolateral surface of the brain are the prefrontal superficial lateral superior, the precentral superficial lateral superior, the central superficial lateral superior, the parietal superficial lateral superior, and the occipital superficial lateral superior veins which drain to the superior sagittal sinus, to bridging veins, and to the falx cerebri. The veins which drain the lateral surface of the brain downwards are the middle superficial cerebral veins, the temporal inferior, occipital inferior, and anastomotic veins. The diameters of these veins were measured at the perforation of the arachnoid membrane and the diameters of the anastomotic veins on their narrowest area. On the medial side of the hemispheres, we divided in precentral superficial superior medial, central superficial medial, parietal superficial medial, occipital superficial medial dorsal veins of the corpus callosum, and internal occipital veins. On the basal surface of the hemispheres, we studied and described the uncal veins and the inferior hemispheric veins. Studied and discussed are also the bridging veins in the course of the inferior cerebral veins, the paracavernous sinuses, and the last course of the veins and their connections with the dura mater or the course inside the dura. Given are besides the numbers of these veins, the area of perforation of the arachnoid membrane, and their width and medical importance.     

Methylene blue plus light mediates 8-hydroxyguanine formation in DNA

Exposure to methylene blue (MB) plus light mediates formation of large levels of 8-hydroxyguanine in DNA. The amount of 8-hydroxy-2'-deoxyguanosine (8-OHdG) present in DNA increased as the amount of MB concentration increased throughout the 2 to 200 microM range studied and was dependent on light exposure. As the time of light exposure increased so did the 8-OHdG content to levels of about 750 8-OHdG/10(5) deoxyguanosine after 15 min of light exposure when MB was at 20 microM. Even though previous research has demonstrated that hydroxyl free radicals formed from a variety of sources mediate 8-OHdG formation in DNA, inclusion of mannitol, superoxide dismutase, catalase, and desferal in the MB plus light experiments demonstrated that these scavengers of oxygen free radical intermediates or precursors caused either no change or an increase in the 8-OHdG content of DNA exposed to MB plus light. These results appear to rule out the direct role of oxygen free radical intermediates in the primary events involved in the MB plus light mediated formation of 8-OHdG in DNA. Oxygen was essential to cause MB plus light mediated 8-OHdG formation in DNA. It was noted that when the reaction was carried out where the deuterium oxide content had been increased to 100%, the amount of 8-OHdG formed in DNA increased about threefold over that observed when comparable reactions were carried out in pure H2O. Use of the singlet oxygen scavenger 2,5-dimethylfuran has yielded variable results on the MB plus light mediated formation of 8-OHdG in DNA. The data taken collectively clearly indicate that MB plus light mediates 8-OHdG formation in DNA. The D2O data and the requirement for oxygen suggest that singlet oxygen may be an intermediate.     

Unusual DNA structures at the integration site of an HIV provirus

Supercoiled pHXBc2 DNA (containing the genome of the human immunodeficiency virus type 1 and human sequences) migrated more slowly than linear DNA in native and ethidium bromide agarose gel electrophoresis at 4.5 volts/cm, suggesting the presence of unusual DNA structures. S1 nuclease analysis of pHXBc2 revealed two S1 hypersensitive sites. Site I was located within a 25 bp direct repeat in host DNA 0.6 kB upstream from the 5' LTR. Site II was mapped 0.2 kB upstream from the vif gene start site. Sequence analysis showed that Site I sequences could assume different unusual DNA structures, whereas sequences at Site II could assume either slipped or H-DNA forms. Unusual DNA structures in host DNA may be associated with active chromatin regions and may favor proviral integration.     

Herbicide effects on planktonic systems of different complexity

Bioassays of different complexity were compared with respect to their capability to predict the environmental impact of the herbicide atrazine in aquatic systems. Acute toxicity tests with Daphnia did not yield meaningful results. Sublethal tests with Daphnia (feeding inhibition, reduction of growth and reproduction) were more sensitive, but effective concentrations of atrazine were still rather high (2 mg/L). A relatively complicated artificial food chain system that incorporated direct and indirect effects on Daphnia yielded significant reduction of daphnid population growth at 0.1 mg/L. Enclosure experiments with natural communities were by far the most sensitive tools. Community responses could be measured at concentrations as low as 1 µg/L and 0.1 µg atrazine/L. At the lowest concentration, however, communities recovered after three weeks. We conclude that in complex systems indirect effects can be more important than direct effects, so that, contrary to the conditions in simple tests, non-target organisms may be the better indicators of herbicide stress to natural communities.     

Structures of acidic O-linked polylactosaminoglycans on human skim milk mucins

O-Linked glycans were isolated from human skim milk mucins or mucin-derived high-molecular weight glycopeptides and fractionated by anion exchange chromatography into neutral and acidic alditols. Major oligosaccharides contained in the acidic fraction were purified by high performance liquid chromatography and structurally characterized by a combination of fast atom bombardment mass spectrometry, methylation analysis and 500 MHz 1H-nuclear magnetic resonance spectroscopy. The structural aspects exhibited by these major species in the acidic fraction resemble those established previously for the neutral oligosaccharides from human skim milk mucins: 1) the size of the alditols varies from tri- to decasaccharides, 2) the core structure is of the ubiquitous type 2, 3) the backbone sequences are of the poly-N-acetyllactosamine type with a particular preponderance of linearly extended GlcNAc beta(1-3)Gal (major) or GlcNAc beta(1-6)Gal units (minor).     

Physiological and biomechanical differences between wheelchair-dependent and able-bodied subjects during wheelchair ergometry

The purpose of this study was to compare the physiological and biomechanical responses of wheelchair-dependent persons (WCD) to able-bodied persons (AB) during manual wheelchair ergometry. Five WCD and five AB performed a discontinuous wheelchair ergometer test starting at 12.8 W at 30 rev.min-1 (57 m.min-1) with increments of 7.0 W at 6-min intervals. Biomechanical data were collected 3.5 min into each stage followed by the collection of physiological data. After the fifth stage, peak oxygen consumption was determined by having the subject work against a resistance of 14.7-19.6 N at 30 rev.min-1. The WCD had significantly higher net mechanical efficiency at 26.7, 33.6 and 40.6 W in comparison to the AB. The WCD had significantly greater shoulder extension at the point of initial wheel contact as measured by the shoulder angle, while the AB had significantly greater shoulder range of motion at all work rates in comparison to the WCD. The results demonstrate that a significant physiological difference exists in the manner by which WCD and AB accomplish wheelchair ergometry. The biomechanical differences between AB and WCD were found to be a prominent factor contributing to the higher mechanical efficiency of WCD over AB. It was concluded that basic physiological and biomechanical differences exist between WCD and AB in manual wheelchair locomotion and that these differences are important considerations to the interpretation of data in wheelchair ergometry studies.     

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.