Growth of animal cells on glass fiber filters and their utilization for biosynthetic analysis

Summary HeLa and L-M cells can be effectively grown directly on glass fiber filters to yield replicate cultures that allow easy analysis of biosynthetic capabilities through measurement of radioactive precursor uptake and incorporation. The glass fiber filters are superior to glass cover slips, growth in scintillation vials, and growth on Millipore filters in the ease of handling during experimental treatment and in the amount of radioactivity incorporated during the labeling period. These parameters are experimentally established and a typical application of the procedure that demonstrates the hydroxyurea inhibition of DNA synthesis is presented. This research was supported by Oklahoma Agricultural Experiment Station Project 1534. This publication is Article J-3334 of the Oklahoma Agricultural Experimental Station.     

Fine structure of the zoospore ofUlothrix belkae with emphasis on the flagellar apparatus

Summary The anterior end of the zoospore ofUlothrix belkae has been examined in detail and is compared toStigeoclonium and other filamentous green algae. The nature of the symmetry of green algal motile cells is discussed and the term, 180° rotational symmetry, is proposed to describe the type of arrangement of anterior end components seen inU. belkae, including the four basal bodies, rootlets and striated fibers. The four microtubular rootlets are cruciately arranged. A striated microtubule-associated component (SMAC) has a periodicity of 6.4 nm and extends with each 2-membered rootlet posteriorly into the cell. One 5-membered rootlet passes very near the eyespot. Phylogeny in green algal motile cells is discussed.     

Hormonal regulation of cotton ovule and fiber growth: Effects of bromodeoxyuridine,AMO-1618 and p-chlorophenoxyisobutyric acid

The effects of 5-bromo-2-deoxyuridine (BUdR, thymidine analogue), AMO-1618 (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride), a growth retardant, and p-chlorophenoxyisobutyric acid (PCIB, an antiauxin) on growth (dry weight increase) and fiber development in unfertilized cotton (Gossypium hirsutum L.) ovules grown in vitro have been studied. BUdR (5 M) causes about 70% inhibition of fiber production, with little effect on ovule growth, if applied during the first 6 d of culture in the presence of GA₃ and IAA. AMO-1618, when used with GA₃ alone, causes only a small reduction in both dry weight and fiber production, but when used with IAA alone reduces both fiber production and dry weight, the effect on the latter being predominant. In the presence of both IAA and GA₃, AMO-1618 causes a small decrease in fiber production but a major decrease in dry weight. PCIB completely inhibits fiber growth but has little effect on dry weight, especially when GA₃ is present. These results indicate that GA₃ mainly promotes ovule growth while IAA is largerly responsible for fiber growth.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride - BUdR 5-bromo-2-deoxyuridine - GA₃ gibberellic acid - IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid - TFU total fiber units     

Parallel measurements of bound calcium and force in glycerinated rabbit psoas muscle fibers

A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca²⁺ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca²⁺-troponin interactions coincident with MgATP-induced force development. Over the free [Ca²⁺] range 6 · 10^?8–1.2 · 10^?5 M the bound Ca²⁺ varied from 0.25 to 1.65 μmol/g protein. The free [Ca²⁺] at half-maximal Ca²⁺ saturation was 2 · 10^?7 M while that a half-maximal force was 5 · 10^?7 M. Half-maximal Ca²⁺ saturation was associated with 20% maximal force. The force-[Ca²⁺] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca²⁺-binding sites is essential for initiation of cross-bridge cycling.     

Differences in the In Vitro and In Vivo 5-Hydroxytryptamine Extraction Performance Among Three Common Microdialysis Membranes

The present study summarizes the results of an in vitro and in vivo comparison of the apparent 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid dialysis performance of three types of membrane frequently used in intracerebral microdialysis experiments. The dialysis fiber types examined were a regenerated cellulose Cuprophan (GF), a proprietary polycarbonate ether (CMA), and a polyacrylonitrile/sodium methallylsulfonate copolymer (HOSPAL). The experiments unexpectedly revealed that the HOSPAL membrane-equipped probes displayed clearly aberrant 5-HT diffusion dynamics compared with GF and CMA probes, demonstrable not only in vitro, but also in in vivo experiments. In vitro, the GF and CMA membrane-equipped probes exhibited maximum relative recovery for 5-HT already in the first 20-min sample, whereas the 5-HT recovery of HOSPAL probes increased in a very slow and protracted manner over a period of a little less than 2 h. The GF and CMA probes further displayed an immediate washout of 5-HT when the probes were subsequently transferred to artificial CSF only-containing medium (no 5-HT), whereas approximately 2 h was required to yield near-total extinction of dialysate 5-HT with the standard HOSPAL probes. In vivo, the rat ventral hippocampal dialysate 5-HT output responses to K+ (100 mM) infusion, to Ca2+ omission, and to systemic 8-hydroxy-2-(di-n-propylamino)tetralin injection were all markedly retarded and blunted when HOSPAL instead of GF membrane-equipped probes were used. However, the 5-hydroxyindoleacetic acid and 3,4-dihydroxyphenylacetic acid extraction in vitro and in vivo were comparable using either of the membrane types.(ABSTRACT TRUNCATED AT 250 WORDS)     

High oxygen transfer rate in a new aeration system using hollow fiber membrane

A new type of bubble aeration column called a hollow fiber membrane (HFM) aeration column was proposed, which was featured in the use of hollow fiber membranes and gave a high bubble density in the column. The value of k(L)a was increased by modifying the membrane surface for making the pore size smaller. The Sauter mean diameter of bubbles (D(vs)) was 2.0 +/- 0.1 mm in the range of the superficial gas velocity from 0.02 m s(-1) to 0.065 m s(-1), while that obtained for the bubbles near the membrane was 811 mum at the superficial gas velocity of 4.0 x 10(-4) m s(-1). The difference was ascribed to the effect of coalescence of bubbles. The value of K(L)a increased in proportion to the superficial gas velocity up to 0.02 m s(-1), and was almost constant above 0.03 m s(-1). The maximum value of k(L)a, 2.5 s(-1), was higher than those of the other aeration columns reported previously. The pneumatic power consumption per unit liquid volume (P(v)) for obtaining the same k(L)a was the smallest in the HFM aeration columns. P(v), for obtaining the same interfacial area of bubbles per liquid volume, was also lower than those for other types of aeration columns. It was suggested from the measurement of bubble diameter that the larger interfacial area generated in the HFM aeration column ascribes to the larger gas holdup than the smaller D(vs). (c) 1992 John Wiley & Sons, Inc.     

Hydrolysis of butteroil by immobilized lipase using a hollow-fiber reactor: part I. lipase adsorption studies

Adsorption of proteins from a crude preparation containing a lipase from Aspergillus niger on microporous polypropylene hollow fibers was studied at six different temperatures. Langmuir isotherms accurately describe the overall adsorption equilibria. Lipase is selectively adsorbed relative to the other proteins in the crude preparation. Hence, immobilization also provides further purification of the lipase. The predictions of the Langmuir model for the change in the specific activity of lipase upon adsorption are consistent with experimental results. The loading capacity of the hollow fibers decreases and the adsorption constant increases as temperature is increased. This effect is more significant in the case of lipolytic activity than it is for the total amount of adsorbed protein. Small, positive enthalpy changes are associated with the adsorption of lipase on these hydrophobic membranes.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

Structural Characteristics of the Short-Tail Fibers of T4 Bacteriophage

The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43-nm fibers were the most abundant structure. During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles. The amino acid composition of the highly purified short-tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.     

2-Deoxyglucose Incorporation in the Cerebellum of Weaver and Nervous Mutant Mice

Abstract: The 2-deoxyglucose autoradiographic method has been used to study activity in cerebellum of the weaver and nervous mutant mice. Patterns of 2-deoxyglucose incorporation into the cerebral hemispheres from weaver and nervous strains did not differ significantly from those of the controls. In the normal cerebellum, 2-deoxyglucose incorporation was maximal in the granular layer, where mossy fibers form synapses with the dendrites of granule cells. In the cerebellum of nervous mice, which lacks Purkinje cells, the incorporation of the 2-deoxyglucose was maximal in the granular layer, but the incorporation into the molecular layer appeared less than in the control. The incorporation into the cerebellum from weaver, which lacks granule cells, was much higher than that of the control, the maximal incorporation being found in the Purkinje cell layer and in cell masses located in the white matter. These data suggest that the heterologous synapses that mossy fibers or climbing fibers form with the cells in the Purkinje cell layer and the cells in the white matter in the weaver cerebellum are functional.