Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

The synovial lining of the rabbit knee: A scanning electron microscopy study of specimens reinforced structurally with tannic acid

Summary Medial and lateral synovial linings of the rabbit knee, structurally reinforced with tannic acid during fixation, were studied in the scanning electron microscope. Low-resolution micrographs revealed, in both linings, gross architecture of four types: accordion-like, lobe-like, fatty areolar, and flattened areas. In high resolution, both cellular and acellular surfaces were recorded. A novel, bubble-like appearance, of unknown nature and origin, accounted for 70% of both linings. No definite correlation between anatomical location, gross type, or microarchitectural pattern was noted.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

The lysis of Gram-negative Alcaligenes eutrophus by enzymes from Cytophaga

Summary The use of Cytophaga lysing enzymes was investigated for the liberation of poly--hydroxybutyrate (PHB) granules from the Gram-negative bacterium Alcaligenes eutrophus. Complete cell lysis was approached within a 60 minute period. Contrary to previous findings for the lysis of Gram-negative bacteria, prior removal of the outer membrane was not essential for enzymic lysis. The destabilisation of the outer membrane by the removal of divalent cations resulted in no significant improvement in the disruption process.     

How do floater male eastern bluebirds benefit by filling vacancies on territories where females already have young?

When male eastern bluebirds (Sialia sialis) are killed or deserttheir mates, replacement males usually take over the territory.We captured 25 eastern bluebird males and held them in captivityto study the behavior of replacement males and their responseto young already present in the nest. Of 17 replacement males,most courted the females, and 23% of experimental females renested,a proportion not different from that of control females. Noreplacement males were infanticidal, and none fully adoptedthe young. Replacement males spent significantly less time onthe territory and defended it significantly less than controlmales. We discuss breeding options available to a floater maleeastern bluebird and conclude that (1) floater male bluebirdsthat fill territory vacancies benefit by gaining breeding opportunitiesin the current breeding season, and (2) indifference to theyoung is the least costly option for replacement male easternbluebirds.     

Factors affecting recurrent shoot multiplication in in vitro cultures of 17- to 20-year-old douglas fir trees

Summary The effects of sucrose concentration, addition of ammonium nitrate, and exposure to N⁶-benzyl-adenine (BA) on multiplication potential with shoots derived from shoot cultures of 17- to 20-yr-old Douglas fir trees [Pseudotsuga menziesii (Mirb.) Franco] were compared. Each of these conditions, when compared independently, affected recurrent shoot multiplication and influenced shoot development, as measured by the abundance of shoot apices. Sucrose concentration was influential, the use of 25 g · liter⁻¹ providing twice the multiplication obtained with 20 g · liter⁻¹, and 14 × that obtained with the 30 g · liter⁻¹ concentration routinely used (tree 11). Ammonium nitrate usage also improved multiplication, a 2.5 times improvement being obtained after incorporation of 100 mg · liter⁻¹ NH₄NO₃ into the medium (tree 33). Shoot cultures were responsive but relatively sensitive to addition of BA, the best improvement in multiplication (5 times) being obtained with brief exposures to 3 mg · liter⁻¹ BA (tree 11). Although shoot cultures were responsive to the conditions investigated, differences in shoot multiplication and development were not displayed for several weeks. It was not possible therefore to repeat all the treatments with more than one genotype; however, when this was possible a genotype-dependent variation in response was evident.     

Effects of high concentrations of secretagogues on the morphology and secretory activity of the pancreas: A role for microfilaments

Summary Cholecystokinin (CCK) and acetylcholine, at concentrations greater than those required for maximal pancreatic enzyme secretion, elicit a submaximal secretory response. The mechanism for this secretagogue-induced unresponsiveness is unknown. Using isolated pancreatic acini of the mouse, we now find that high concentrations of secretagogues also induce a profound alteration in acinar morphology, characterized by the formation of spherical protrusions on the basal surface of the cells. Since both the determination of cell shape and exocytosis may involve calcium and contractile proteins, we used a calcium-free medium and cytochalasin B (CB) to evaluate the importance of a contractile mechanism in the secretory and morphological effects of high concentrations of CCK-octapeptide (CCK₈). Incubation in a calcium-free medium partially blocked CCK-induced unresponsiveness, but brought about dissociation of the acini. CB at a concentration of 3 g/ml caused the disappearance of apical microfilaments and, most strikingly, completely prevented the morphological alteration induced by CCK₈. Furthermore, CB converted the biphasic dose-response curve for CCK₈-induced amylase release to a monophasic shape, such that the amylase release stimulated by a high concentration of CCK₈ (10 nM) was augmented. It is concluded, therefore, that a contractile process involving microfilaments may mediate secretagogue-induced unresponsiveness in pancreatic acinar cells.     

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Fifteen nitrate assimilation-deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO₃^? but able to grow on NO₂^?, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO₃^? or NO₂^?, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen-nitrate reductase in the presence of nitrite reductase is described.