Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator

Summary Cl transport in apical membrane vesicles derived from bovine tracheal epithelial cells was studied using the Cl-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl) quinolinium. With an inwardly directed 50 mM Cl gradient at 23°C, the initial rate of Cl entry (J _Cl) was increased significantly from 0.32±0.12 nmol · sec^–1 · mg protein^–1 (mean±sem) to 0.50±0.07 nmol · sec^–1 · mg protein^–1 when membrane potential was changed from 0 to +60 mV with K/valinomycin. At 37°C, with membrane potential clamped at 0 mV, there was a 34±7% (n=5) decrease inJ _Cl from a control value of 0.37±0.03 nmol · sec^–1 · mg protein^–1 upon addition of 0.2mm diphenylamine-2-carboxylate. The following did not alterJ _Cl significantly (J _Cl values gives as percent change from control): 50mm cis Na (–1±5%), 0.1mm furosemide (–3±4%), 0.1mm furosemide in the presence of 50mm cis Na (–5±2%), 0.1mm H₂DIDS (–18±9%), a 1.5 pH unit inwardly directed H gradient (–7±7%), and 0.1mm H₂DIDS in the presence of a 1.5 unit pH gradient (4±18%). With inward 50mm anion gradients, the initial rates of Br and I entry (J _Br andJ ₁, respectively) were not significantly different fromJ _Cl.J _Cl was a saturable function of Cl concentration with apparentK _d of 24mm and apparentV _max of 0.54 nmol · sec^–1 · mg protein^–1. Measurement of the temperature dependence ofJ _Cl yielded an activation energy of 5.0 kcal/mol (16–37°C). These results demonstrate that Cl transport in tracheal apical membrane vesicles is voltage-dependent and inhibited by diphenylamine-2-carboxylate. There is no significant contribution from the Na/K/2Cl, Na/Cl, or Cl/OH(H) transporters. The conductive pathway does not discriminate between Cl, Br, and I and is saturable. The low activation energy supports a pore-type mechanism for the conductance.     

Na−K−2Cl cotransport in winter flounder intestine and bovine kidney outer medulla: [3H] bumetanide binding and effects of furosemide analogues

Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC₅₀ values) and order of potency of these derivatives were benzmetanide, 5×10^–8 m> bumetanide 3×10^–7 m>piretanide 3×10^–6 m>furosemide 7×10^–6 m> amino piretanide 1×10^–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [³H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK _d and number of binding sites (n) were: bovine kidney,K _d =1.6×10^–7,n=10.5 pmol/mg protein; winter flounder intestine,K _d 1.2×10^–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K _d =5.3×10^–7,n=20.4 pmol/mg protein. The estimatedK _d for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10^–7 m. The similarity in magnitudes of IC₅₀ andK _d for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK _d andn were lower at the higher Cl concentration (5mm Cl:K _d =5.29×10^–7 m,n=20.4 pmol/mg protein; 100mm Cl:K _d =2.3×10^–7 m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl.     

Plant regeneration through somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l^–1 N⁶-benzyladenine (BA) and 0–2.0 mg l^–1 -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l^–1 BA and 2.0 mg l^–1 2,4-D. Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l^–1 BA and 0–0.4 mg l^–1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l^–1 BA and 0.2 mg l^–1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l^–1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos.     

Induction of haploid callus and embryogenesis in in vitro cultured anthers of mulberry (Morus indica)

Anthers of Morus indica L., with microspores at the uninucleate stage were cultured; and the influence of temperature and kinetin pretreatment on induction of androgenic calluses was examined. The effects of various pretreatments revealed that 24 h cold pretreatment increased the percentage of cultures inducing callus. First microspore division was observed after 16 to 20 days of culture. Th anthers split and developed embryogenic calluses on MB medium supplemented with NAA (0.5 mg l^–1 and BA (1.0 mg l^–1)) using 8% sucrose. Rhizogenesis was induced on medium supplemented with NAA and BA (each 0.5 mg l^–1) with reduced myo-inositol (75 mg l^–1). Cytological study of induced roots confirmed the haploid nature of calluses. Different type of embryos were initiated upon transfer of calluses to medium supplemented with NAA, BA (each 0.5 mg l^–1), 2,4-d (1.0 mg l^–1) and PVP (600 mg l^–1). These embryoids further developed roots on removal of 2,4-d from the medium and developed precociously without developing cotyledons and formed elongated shoots.Abbreviations BA 6 benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin: Acetic acid: Alcohol - GA₃ gibberellic acid - IBA indole-3-butyric acid - MB modifed Bourgin (Qian et al., 1982) - NAA 1-naphthalene acetic acid - PVP polyvinylpyrrolidone - RFS-135 rainfed selection 135 - SE standard error     

Voltage dependence of Na/K pump current inXenopus oocytes

Summary Stage V and VI (Dumont, J.N., 1972.J. Morphol. 136:153–180) oocytes ofXenopus laevis were treated with collagenase to remove follicular cells and were placed in K-free solution for 2 to 4 days to elevate internal [Na]. Na/K pump activity was studied by restoring the eggs to normal 3mm K Barth's solution and measuring membrane current-voltage (I–V) relationships before and after the addition of 10 m dihydroouabain (DHO) using a two-microelectrode voltage clamp. Two pulse protocols were used to measure membraneI–V relationships, both allowing membrane currents to be determined twice at each of a series of membrane potentials: (i) a down-up-down sequence of 5 mV, 1-sec stair steps and (ii) a similar sequence of 1-sec voltage pulses but with consecutive pulses separated by 4-sec recovery periods at the holding potential (–40 mV). The resulting membraneI–V relationships determined both before and during exposure to DHO showed significant hysteresis between the first and second current measurements at each voltage. DHO difference curves also usually showed hysteresis indicating that DHO caused a change in a component of current that varied with time. Since, by definition, the steady-state Na/K pumpI–V relationship must be free of hysteresis, the presence of hysteresis in DHO differenceI–V curves can be used as a criterion for excluding such data from consideration as a valid measure of the Na/K pumpI–V relationship. DHO differenceI–V relationships that did not show hysteresis were sigmoid functions of membrane potential when measured in normal (90mm) external Na solution. The Na/K pump current magnitude saturated near 0 mV at a value of 1.0–1.5 A cm^–2, without evidence of negative slope conductance for potentials up to +55 mV. The Na/K pump current magnitude in Na-free external solution was approximately voltage independent. Since these forward-going Na/K pumpI–V relationships do not show a region of negative slope over the voltage range –110 to +55 mV, it is not necessary to postulate the existence of more than one voltage-dependent step in the reaction cycle of the forward-going Na/K pump.     

Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium

Summary Internal perfusion ofHelix neurons with a solution containing potassium aspartate, MgCl₂, ATP, and HEPES causes the calcium-activated potassium current (I _K(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca⁺⁺ is strongly buffered to 10^–7 m by including 0.5mm EGTA and 0.225mm CaCl₂ in the internal solution,I _K(Ca) remains constant for up to 3 hours of perfusion. In cells whereI _K(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10^–7 m free Ca⁺⁺ solution produces increases inI _K(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10^–6 m free Ca⁺⁺,I _K(Ca) is low and does not change with perfusion. These results lead us to conclude thatI _K(Ca) is stable in perfusedHelix neurons and that the apparent loss ofI _K(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (I _Ca) provides the Ca⁺⁺ which activatesI _K(Ca) during a depolarizing pulse,I _Ca is also stable in perfused cells when free intracellular Ca⁺⁺ is buffered.Perfusion with 1 m calmodulin (CaM) produces no effect onI _K(Ca) with either 10^–7 or 10^–6 m free internal calcium. Inhibiting endogenous CaM by including 50 m trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect onI _K(Ca) with 10^–7 m free internal calciu. It is concluded that CaM plays no role inI _K(Ca) activation.     

DNA replication sequence of human chromosomes in blood cultures

Summary The pattern of labelling over the chromosomes, the chronology of chromosome duplication and the duration of the S and G ₂ periods in the leukocytes from 6 normal females and 5 normal males, have been studied by using a combination of pulse and continuous tregtments with thymidine-H³. According to the criteria used to analyse the results it is suggested that the S period begins 15 to 20 hours and finishes 5 to 3 hours before the cells reach the metaphase stage. The S period could be subdivided into the four phases S₁ to S₄.The first chromosomes to replicate were Nos. 1, 3, 5 and X followed by the Nos. 2, 4 and several chromosomes of groups 6–12, 13–15 and 19–20. Later the pairs 16, 17, 18 and the chromosomes of group 21–22 replicated. Chromosome Y in the male was the last to replicate, beginning its duplication when all the other chromosomes had reached the intermediate S stage.The earliest chromosomes to finish the duplication were Nos. 19, 20 and 21 followed by Nos. 16, 17, 18, 22 and the chromosomes of group 13–15. Afterward and at about the same time the replication of pairs 2, 4, 6, 8, the X and Y chromosomes in the male and one X chromosome in the female concluded. The other X chromosome in the female was the last to end its duplication appearing totally labelled until the final stage of the S period.Replication of the long and medium size chromosomes begins at localised regions, then extends over the total length of the chromosome and at the end of the S stage takes place only in small zones different from those replicating early.Asynchrony between homologous chromosomes was observed at the beginning and at the end of the S period.     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Anaerobic degradation of p-cresol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of p-cresol under denitrifying conditions by a bacterial consortium was studied in batch and continuous cultures. Concentrations up to 3 mm were degraded within 5–6 days with 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde and 4-hydroxybenzoate as intermediates. Steady states could be maintained at only one dilution rate, D=0.04 h^–1. A further increase in the dilution rate to 0.0 8 h^–1 resulted in culture wash-out. An estimation of the Saturation constant was made (<1 mg/l), taking the maximum specific growth rate as 0.045 h^–1, thus yielding a value of 0.125 mg p-cresol/l. Correspondence to: N. Khoury     

Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was –65.2 mV and the slope resistance 150 to 750 M. Pulse-depolarization from a holding voltage of –80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at –25 mV and was half-maximal at –8 mV. Steady-state inactivation was half-maximal at –67 mV and complete at –50 mV. Peak Na current averaged –0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 m in the pipette) caused reversible depolarization (to –40 to –20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP--S in the pipette caused slow depolarization to –40 to –20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

Modeling available 2,4-dichlorophenoxyacetic acid in a tissue culture medium containing activated carbon

Clonal propagation of high-value forest trees by somatic embryogenesis can help meet industry needs for uniform and high quality raw materials. Low embryogenic tissue initiation frequencies for loblolly pine (Pinus taeda L.) pose a limitation in work towards commercialization of this technology. At the time our research began most work on somatic embryo culture initiation in loblolly pine reported success in the range of 1–5%. Activated carbon (AC) has been reported to improve many tissue culture systems including embryogenic tissue initiation in Douglas-fir. To improve initiation frequencies in loblolly pine, the development of an AC-containing system was explored. In order to better understand the availability of 2,4-dichlorophenoxyacetic acid (2,4-D) in initiation medium, we tracked media surface concentrations of free or available 2,4-D. Media containing 1/2 modified P6 salts, 1.5% maltose, 2% myo-inositol, case amino acids, glutamine, vitamins, and 0.4% Gelrite were modified to include 0.625 – 2.5 g l^–1 of activated carbon (Sigma C-9157, acid washed) and 110 –440 mg l^–1 2,4-D. Adsorption and availability of 2,4-D in AC-containing medium was tracked by C¹⁴ labeled 2,4-D present in surface moisture absorbed into filter paper. High correlations were found between–available 2,4-D and time when AC and initial 2,4-D concentrations were held constant,–available 2,4-D and AC concentration when initial added 2,4-D and time were held constant, and–available 2,4-D and initial 2,4-D when AC and time were held constant.All of these relationships were exponential, not linear. Multiple regression models inputting initial 2,4-D added to medium in mg l^–1, activated carbon added to medium in %, and time in days, were able to explain 85–88% of the variability in available 2,4-D. These models can be used to achieve target levels of available 2,4-D by adjustment of initial 2,4-D levels or AC content.     

Some effects of trinitrocresolate and valinomycin on Na and K transport across thin lipid bilayer membranes: A steady-state analysis with simultaneous tracer and electrical measurements

Summary This paper describes the effect of trinitrocresolate anions (TNC^–) on the electrical conductance (G _m ), and tracer-measured unidirectional Na and K fluxes (M _Na andM _K) across bilayers formed from sheep red cell lipids dissolved in decane. In the absence of TNC^–, typical low conductances were observed, while the cation fluxes were too low to measure by our techniques (<10^–12 moles cm^–2 sec^–1). In the presence of TNC^– (10^–2 m),G _m increased and TNC^– was the main charge carrier in the system. The cationic fluxes were also much increased, but the membranes showed no significant selectivity between K and Na. Furthermore, the Na and K fluxes were at least two orders of magnitude larger than the ionic fluxes calculated fromG _m . Thus, almost all of the K and Na transport across the membrane in the presence of TNC^– is electrically silent and is probably carried out as KTNC and NaTNC ion pairs.In the presence of valinomycin (10^–6 m) and no TNC^–, both the ion fluxes andG _m were 10³ times larger in KCl than in NaCl, thus exhibiting the characteristic high selectivity of valinomycin for K over Na. In the presence of both valinomycin (10^–6 m) and TNC^– (10^–2 m), this selectivity disappeared in that bothG _m andM _Na in the NaCl system were similar to the respective values in the KCl system. Even under these conditions, most of the Na is still transported by a process which does not carry charge.BothG _m andM _x increased alike and monotonically with increasing temperature over the range 7 to 30°C. In the absence of TNC^– the enthalpies of activation were invariably higher in KCl than in NaCl. Addition of TNC^– produced equal enthalpies of activation for both Na and K containing systems suggesting a common, temperature-dependent, ratedetermining step in charge transfer and the electrically silent cation fluxes.     

Kinetic Characterization of Apical <Emphasis Type="SmallCaps">D</Emphasis>-Fructose Transport in Chicken Jejunum

In mammals, D-fructose transport takes place across the brush-border membrane of the small intestine through GLUT5, a member of the facilitative glucose transporter family. In the present paper, we describe and characterize for the first time the apical transport of D-fructose in chicken intestine. Brush-border membrane vesicles (BBMV) were obtained from jejunum of 5- to 6-wk-old chickens. D-Fructose uptake by BBMV from chicken jejunum comprises a saturable component and a simple diffusion process. The maximal rate of transport (V_max) for D-fructose was 2.49 nmol·(mg prot)^–1·s^–1, the Michaelis constant (K_m) was 29 mM, and the diffusion constant (K_d) was 25 nl·(mg prot)^–1·s^–1. The apical transport of D-fructose was Na⁺-independent, phlorizin-, phloretin-, and cytochalasin B-insensitive, and did not show cis-inhibition by D-glucose or D-galactose. These properties, together with the detection of specific GLUT5 mRNA, indicate the presence of a low-affinity high-capacity GLUT5-type carrier in the chicken jejunum, responsible for the entry of D-fructose across the brush-border membrane of enterocytes.     

Metabolism of d(+)-carnitine by Escherichia coli

Summary Escherichia coli 044 K74 grown under anaerobic conditions in the presence of l(–)-carnitine is able to convert d(+)-carnitine into the l(–)-enantiomer. This activity is repressed by electron acceptors such as oxygen and nitrate as well as by glucose. d(+)-Carnitine shows no effect on the induction or repression of the corresponding enzyme or enzyme system. Resting cells of E. coli 044 K74 were used for the formation of l(–)-carnitine from d(+)-carnitine. The maximum obtained yield was 50%. -Butyrobetaine was formed as a by-product. Offprint requests to: H. Jung     

Shoot regeneration,re-flowering and post flowering survival in bamboo inflorescence culture

In vitro grown inflorescences of Bambusa edulis were used to investigate the process of vegetative shoot growth in detail. The findings revealed that auxins and ACC could be significant growth regulators in this process. Overall, auxins [NAA, indolebutyric acid (IBA), and 2,4-dichlorophenoxyacetic acid (2,4-D)] induced inflorescences to grow vegetative shoots. However, the efficiency of shoot regeneration varied. A greater percentage (27.3–34.5) of inflorescences in the 5 mg l⁻¹ NAA, 10 mg l⁻¹ NAA, and 1 mg l⁻¹ 2,4-D treatments formed more vegetative shoots than those exposed to other treatments. IBA promoted shoot regeneration less effectively than NAA and 2,4-D. Fifty percent of regenerated vegetative shoots flowered after 2 months when the medium was supplemented with 5 mg l⁻¹ NAA. All shoots that received 1 mg l⁻¹ 1-amino-cyclopropane-1-carboxylic acid (ACC) flowered in 5 mg l⁻¹ NAA medium. Rooted plantlets were used to examine their survival following in vitro flowering. All plantlets with vegetative shoots, even those with inflorescences, survived and grew.     

Role of vacuolar adenosine triphosphatase in the regulation of cytosolic pH in hepatocytes

The responses of the cytosolic pH of hepatocytes in suspension to agents affecting the activity of vacuolar adenosine triphosphatase (V-ATPase) and Na/H exchange have been studied. Changes of cytosolic pH were determined both with dual-wavelength excitation (500/440 nm) of the fluorescence of 2,7-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein and from the distribution of ¹⁴C-dimethyloxazolidinedione; both methods gave very similar results. Changes of vesicular pH were determined by comparing the fluorescence of fluorescein isothiocyanate-dextran and rhodamine B isothiocyanate-dextran taken up by endocytosis. Nitrate, which inhibits V-ATPase in isolated organelles, induced a concentration-dependent acidification of the cytosol and alkalinization of vesicles, with maximal effects at 25–37.5 mm in each case, indicating that V-ATPase contributes to removal of cytosolic protons. On continued exposure to nitrate, the acidification underwent an amiloride-inhibitable reversal. At the higher concentrations of NO ₃ ^– , both cytosolic acidification and vesicular alkalinization were reduced or absent. Bafilomycin A₁ caused alkalinization of vesicular pH; cytosolic acidification was not observed, possibly because of other ionic exchanges. Recovery of cytosolic pH from an acid load (2 min exposure to 5% CO₂) was sensitive to both 25 mm NO ₃ ^– and to ouabain. The pH dependence of the nitrate effect was tested with media of different pH; the activity was negligible at cytosolic pH 6.2 and rose to a maximum at cytosolic pH 7.3. Treatment of hepatocytes with 0.5–1.0 mm ouabain resulted in an initial alkalinization (0.5–2 min duration) of the cytosol, followed by a spontaneous reversal and, on occasion, further acidification. The alkalinization was blocked by 25 mm NO ₃ ^– , but not by 25 mm gluconate. The results suggest that the cytosolic alkalinization is caused by a stimulation of H⁺ uptake by V-ATPase activity. We conclude that V-ATPases make an important contribution to the regulation of the cytosolic pH of hepatocytes.This work was supported in part by National Institutes of Health B.R.S. Grant 507 RR05417 to Temple University.     

Fermentation of glycerol to 1,3-propanediol in continuous cultures of Citrobacter freundii

The conversion of glycerol to 1,3-propanediol by Citrobacter freundii DSM 30040 was optimized in single- and two-stage continuous cultures. The productivity of 1,3-propanediol formation was highest under glycerol limitation and increased with the dilution rate (D) to a maximum of 3.7 g·l^–1·h^–1. Glycerol dehydratase seemed to be the rate-limiting step in 1,3-propanediol formation. Conditions for the two-stage fermentation process were as follows: first stage, glycerol limitation (250mM), pH 7.2, D=0.1 h^–, 31° C; second stage, additional glycerol, pH 6.6, D=0.05 h^–1, 28° C. Under these conditions 875mM glycerol were consumed, the final 1,3-propanediol concentration was 545mM, and the overall productivity 1.38 g·1^–1·h^–1. Correspondence to: G. Gottschalk     

Voltage-dependent calcium and potassium conductances in striated muscle fibers from the scorpion,Centruroides sculpturatus

Ionic currents responsible for the action potential in scorpion muscle fibers were characterized using a three-intracellular microelectrode voltage clamp applied at the fiber ends (8–12°C). Large calcium currents (I _Ca) trigger contractile activation in physiological saline (5 mm Ca) but can be studied in the absence of contractile activation in a low Ca saline (2.5 mm). Barium (Ba) ions (1.5–3 mm) support inward current but not contractile activation.Ca conductance kinetics are fast (time constant of 3 msec at 0 mV) and very voltage dependent, with steady-state conductance increasing e-fold in approximately 4 mV. Half-activation occurs at –25 mV. Neither I _Ca nor I _Ba show rapid inactivation, but a slow, voltage-dependent inactivation eliminates I _Ca at voltages positive to –40 mV. Kinetically, scorpion channels are more similar to L-type Ca channels in vertebrate cardiac muscle than to those in skeletal muscle.Outward K currents turn on more slowly and with a longer delay than do Ca currents, and K conductance rises less steeply with voltage (e-fold change in 10 mV; half-maximal level at 0 mV). K channels are blocked by externally applied tetraethylammonium and 3,4 diaminopyridine.This work was supported by a grant from the NIH (NS-17510) to W.F.G. and a NRSA award to T.S. (GM-09921).     

Two type II keratin genes are localized on human chromosome 12

Summary Human genomic DNA containing two type II keratin genes, one coding for keratin 1 (K1, a 68-kD basic protein) and another closely linked type II gene 10–15 kb upstream (K?, gene product unknown), was isolated on a single cosmid clone. EcoRI restriction fragments of the cosmid were subcloned into pGEM-3Z, and specific probes comprising the C-terminal coding and 3 noncoding regions of the two genes were constructed. The type II keratin genes were localized by in situ hybridization of the subcloned probes to normal human lymphocyte chromosomes. In a total of 70 chromosome spreads hybridized with the K? probe (gHK?-3, PstI, 800 bp), 36 of the 105 grains observed were on chromosome 12, and 32 of these were clustered on the long arm near the centromere (12q11–13). In 100 labeled metaphases hybridized with the K1 probe (gHK1–3, BamHI-PstI, 2100 bp), 53 grains localized to chromosome 12 and 46 of these were found in the same region (q11–13). Therefore, both the gene for human keratin 1, a specific marker for terminal differentiation in mammalian epidermis, and another closely linked unknown type II keratin gene (K?, 10–15 kb upstream of K1) are on the long arm (q11–13) of human chromosome 12.