Quantitation of mitochondrial injury in cultured rat heart endothelioid cells with nitroblue tetrazolium

Synopsis A sensitive method is presented for measurement of changes in the permeability of mitchondria in cultured cells. Rat heart endothelioid cells were used to determine the penetration rate of nitroblue tetrazolium (NitroBT) or other reactants into mitochondriain situ. Nitroblue formazan, produced as a consequence of succinate dehydrogenase activity in the mitochondria, was eluted and measured with a spectrophotometer. Prior injury of cells with hypo-osmolar solutions increased the rate of formazan production. Several methods are described or suggested for the statistical analysis of the data.     

Demonstration of immunoreactive forms of erythrocyte protein 4.2 in nonerythroid cells and tissues

Protein 4.2 is a major component of the erythrocyte membrane cytoskeleton. Here we show that immunoreactive forms of human (Mr 72,000) and pig (Mr 75,000) protein 4.2 are also associated with the plasma membrane of various nonerythroid cells and tissues, such as platelets, brain, and kidney. Protein 4.2 can be extracted from platelet membranes under the same conditions (pH 11, 1 M KI, 1 M urea) which are required to extract protein 4.2 from the erythrocyte plasma membrane. The demonstration of protein 4.2 in nucleated cells that contain also several other proteins of the erythrocyte membrane cytoskeleton indicates some general principles underlying the molecular construction of the plasma membrane in erythrocytes and nonerythroid cells.     

Joining of linear plasmid DNA is reduced and error-prone in Bloom''s syndrome cells.

A linearized, replicating, shuttle vector plasmid, pZ189, was used to measure in vivo DNA joining ability of cells from patients with the cancer-prone, immunodeficient, chromosome breakage disorder, Bloom's syndrome (BS). The BS cell lines we studied were reported to contain reduced in vitro activity of DNA ligase I. We assessed in vivo joining ability by transfecting linear plasmids with overlapping or blunt ends (produced by EcoRI or StuI) into BS and normal fibroblast or lymphoblast host cells and measuring the amount of re-joined, replicated plasmids by their ability to transform bacteria. With plasmids having either overlapping or blunt ends we found a 1.3- to 3-fold lower (P less than 0.05) joining efficiency in BS cells than in the normal cells. The mutation frequency of the recovered plasmids was measured by screening for function of the suppressor tRNA contained in pZ189, for plasmid size, for presence of restriction sites, or by DNA sequencing. The spontaneous mutation frequency with the circular plasmid was 0.05-0.08% with both BS cell lines, values 2- to 21-fold higher (P less than 0.03) than with the normal cell lines. The mutation frequency with the linear plasmid passaged through both BS cell lines was 21-52%, values 1.4- to 5.4-fold higher (P less than 0.001) than with the normal lines. Detailed analysis of 210 recovered plasmids revealed an increase (P less than or equal to 0.001) in deletions, insertions or complex mutations at the joining sites, and in point mutations with the EcoRI cut plasmid with the BS cells in comparison to the normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 M PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 M PAA) and with a 16-h light/8-h dark photoperiod (500 M PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 M PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 M as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 M PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 M.Paper No. 88514 of the Journal Series of the Idaho Agricultural Experiment Station, Moscow, Idaho, USA.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 μM PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 μM PAA) and with a 16-h light/8-h dark photoperiod (500 μM PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 μM PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 μM as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 μM PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 μM.     

Responses of plasma human atrial natriuretic factor to high intensity submaximal exercise in the heat

No data exists regarding responses of human atrial natriuretic factor (ANF) to exercise in the heat. The purpose of this study was to examine the responses of plasma ANF to high intensity submaximal (71% +/- 0.9 VO2max) exercise in the heat over an eight day acclimation period. Fourteen healthy males volunteered to participate in the study. Subjects performed intermittent exercises on a treadmill (0% grade) during 50 min of each 100 min trial in an environmental chamber maintained at 41.2 +/- 0.5 degrees C, 39.0 +/- 1.7% relative humidity. Blood was obtained from an antecubital vein after standing 20 min in the heat prior to exercise, and immediately after exercise. Measures were compared on days 1, 4 and 8. ANF did not change pre- to post-exercise nor did it change over the eight day heat acclimation period despite other heat acclimation adaptations. Conversely, plasma aldosterone (ALDO), renin activity (PRA) and cortisol (COR) all increased (p less than 0.05) pre- to post-exercise on each day but again no changes were observed over the eight day period. These data support that ANF may not increase when ALDO and PRA increases are observed.     

Studies of feedback suppression of bile salt synthesis in the bile-fistula rat

We have previously reported that intravenous infusion of taurocholate at 10 mumol (100 g.hr) into bile-fistula rats suppressed bile salt synthesis by 85% (Pries et al. 1983. J. Lipid Res. 24: 141-146). Recently, however, infusion rates twice this high have been reported not to suppress synthesis (Davis et al. 1984. Falk Symposium 42. MTP Press Ltd., Boston. 37-45). Because the only major difference in design of these two studies was supplementation with sodium bicarbonate to replace biliary losses induced by bile salt choleresis, we have repeated our studies with and without bicarbonate supplementation. Without bicarbonate, as before, we found suppression of synthesis during infusion of taurocholate at 10 mumol/(100 g.hr). With bicarbonate, no suppression of synthesis occurred at these infusion rates. These data indicate that bicarbonate supplementation is essential when testing physiological effects of infused bile salt in the bile-fistula rat.