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1.
Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic
callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l−1 (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l−1 (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus
had high levels of Na+, sugar, free amino acids, and proline but a slight decline was recorded in K+ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0
mg l−1 (9.0 μM) 2,4-D+0.5 mg l−1 (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l−1 (0.1 μM) α-naphthaleneacetic acid +0.1 mg l−1 (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing
0.2% (w/w) NaCl. 相似文献
2.
Isoform-Specific Function and Distribution of Na/K Pumps in the Frog Lens Epithelium 总被引:3,自引:1,他引:2
Epithelial cells from the anterior and equatorial surfaces of the frog lens were isolated and used the same day for studies
of the Na/K ATPase. RNase protection assays showed that all cells express α1- and α2-isoforms of the Na/K pump but not the α3-isoform, however the α2-isoform dominates in anterior cells whereas the α1-isoform dominates in equatorial cells. The whole cell patch-clamp technique was used to record functional properties of the
Na/K pump current (I
P
), defined as the current specifically inhibited by dihydro-ouabain (DHO). DHO-I
P
blockade data indicate the α1-isoform has a dissociation constant of 100 μm DHO whereas for the α2-isoform it is 0.75 μm DHO. Both α1- and α2-isoforms are half maximally activated at an intracellular Na+-concentration of 9 mm. The α1-isoform is half maximally activated at an extracellular K+-concentration of 3.9 mm whereas for the α2-isoform, half maximal activation occurs at 0.4 mm. Lastly, transport by the α1-isoform is inhibited by a drop in extracellular pH, which does not affect transport by the α2-isoform. Under normal physiological conditions, I
P
in equatorial cells is approximately 0.23 μA/μF, and in anterior cells it is about 0.14 μA/μF. These current densities refer
to the area of cell membrane assuming a capacitance of around 1 μF/cm2. Because cell size and geometry are different at the equatorial vs. anterior surface of the intact lens, we estimate Na/K pump current density per area of lens surface to be around 10 μA/cm2 at the equator vs. 0.5 μA/cm2 at the anterior pole.
Received: 17 May 2000/Revised: 11 August 2000 相似文献
3.
A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially
for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the
11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic
acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition
of caffeic acid formation was caused by 10−4Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10−3M glyphosate concentration, while 10−3M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation
is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures. 相似文献
4.
Barrière H Rubera I Belfodil R Tauc M Tonnerieux N Poujeol C Barhanin J Poujeol P 《The Journal of membrane biology》2003,193(3):153-170
Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I– > Br– > Cl– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K+ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl– and K+ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures. 相似文献
5.
The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10−6
m l-lysine and 1500∶1 for 10−6
m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically.
The influx is saturable with an apparentK
m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was
13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino
acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed
a powerful “trans” inhibitory effect on the uptake of amino acids. 相似文献
6.
This article describes conditions to optimize the yield of viable protoplasts from callus tissue of Asparagus densiflorus cv. Sprengeri and their subsequent regeneration into plantlets. Callus tissue was initiated by culturing spear sections (5–7
mm) on Murashige and Skoog (MS) medium supplemented with 0.8% (wt/vol) Bacto agar, 3% (wt/vol) sucrose, 0.5 mg/l each of nicotinic
acid, pyridoxine-HCl, and thiamine-HCl, 1 mg/l p-chlorophenoxyaceticacid (pCPA) and 1 mg/l 6-benzylaminopurine (BAP). The maximum protoplast yield was obtained in a mixture
of 1% (wt/vol) Cellulysin, 0.8% (wt/vol) Rhozyme HP 150 and 0.3% (wt/vol) Macerase, dissolved in cell protoplast wash salt
solution with 7 mm CaCl2
.2H2O, 3 mm MES, 0.6 m glucose, and 0.1 m mannitol. First divisions were observed after 3–4 days of initial culture. The plating efficiency was highest (7.8%) in half-strength
MS semisolid medium containing 1 g/l glutamine, 0.6 m glucose, 0.1 m mannitol, 0.5 mg/l folic acid, 0.05 mg/l biotin, 2 mg/l ascorbic acid, 1 mg/l α-naphthaleneacetic acid, 0.5 mg/l zeatin, and 0.1% (wt/vol) Gelrite. Protoplast-derived microcolonies and microcalli were
cultured on the same medium on which the primary callus culture was initiated. After 10–12 weeks, calli were transferred to
shoot regeneration medium containing MS salts, 1 mg/l BAP, 0.5 mg/l pCPA and 0.2% Gelrite. Shoots (3–4 cm) were then transferred
to MS rooting medium with 2 mg/l indole-3-butyric acid, and 0.2% Gelrite. Plantlets were obtained within 4–5 weeks.
Received: 9 August 1995 / Revision received: 27 June 1997 / Accepted: 17 July 1997 相似文献
7.
The effect of transnitrosation intermediate between S-nitroso-N-acetylcysteine (NACysNO) and cysteine on the growth of vgb-bearing Enterobacter aerogenes was investigated using three parameters: the ratio of the specific growth rates, the inhibition zone, and α-amylase synthesis for the culture exposed to stressors
to that of the same stressor-free cultures. The effect of NACysNO/cysteine on the growth of Enterobacter strains was distinctive as compared with the CysNO, NACysNO, and their combination. At a higher concentration (2 mM), the extents of inhibition based on the μNACysNO/cysteine/μno stress ratio for these cultures were 57%, 62%, and 68% for VHb-expressing, parental, and pUC9-harboring cells, respectively. The
inhibition caused by 2 mM NACysNO in the presence of 1 mM cysteine in all bacterial strains was almost twofold that achieved by NACysNO alone. Based on the diameter of the inhibition
zone and α-amylase productivity, the four compounds (NACysNO/Cysteine, CysNO, NACysNO, and their combinations) affected the
E. aerogenes strains in a concentration-dependent and negative manner. This negative effect was lower in vgb-bearing than vgb-lacking strains. Thus, sulfur-to-sulfur transnitrosation was an efficient NO release and significantly (P < 0.05) affects the growth of Enterobacter strains, to a lesser extent in vgb-bearing strains. 相似文献
8.
d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means
of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na+-dependent with K
m
= 5 μm and V
max= 0.7 nmoles · min−1· mg protein−1. Influx is sigmoidal as f[Na+] with Na+
K
m
∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold
increase in V
max, with no change in K
m
. At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the ΔμNa+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response
still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms.
mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are
expressed in these cells.
Received: 11 January 2001/Revised: 26 March 2001 相似文献
9.
Díez-Sampedro A Lostao MP Wright EM Hirayama BA 《The Journal of membrane biology》2000,176(2):111-117
Using cotransporters as drug delivery vehicles is a topic of continuing interest. We examined glucose derivatives containing
conjugated aromatic rings using two isoforms of the Na+/glucose cotransporter: human SGLT1 (hSGLT1) and pig SGLT3 (pSGLT3, SAAT1). Our studies indicate that there is similarity
between SGLT1 and SGLT3 in the overall architecture of the vestibule leading to the sugar-binding site but differences in
translocation pathway interactions. Indican was transported by hSGLT1 with higher affinity (K0.5 0.06 mm) and 2-naphthylglucose with lower affinity (K0.5 0.5 mm) than α-methyl-d-glucopyranoside (αMDG, 0.2 mm). Both were poorly transported (maximal velocities, I
max
, 14% and 8% of αMDG). Other compounds were inhibitors (K
i
s 1–13 mm). In pSGLT3, indican and 2-naphthylglucose were transported with higher affinity than αMDG (K0.5s 0.9, 0.2 and 2.5 mm and relative I
max
s of 80, 25 and 100%). Phenylglucose and arbutin were transported with higher I
max
s (130 and 120%) and comparable K0.5s (8 and 1 mm). Increased affinity of indican relative to αMDG suggests that nitrogen in the pyrrole ring is favorable in both transporters.
Higher affinity of 2-naphthylglucose for pSGLT3 than hSGLT1 suggests more extensive hydrophobic/aromatic interaction in pSGLT3
than in hSGLT1. Our results indicate that bulky hydrophobic glucosides can be transported by hSGLT1 and pSGLT3, and discrimination
between them is based on steric factors and requirements for H-bonding. This provides information for design of glycosides
with potential therapeutic value.
Received: 18 February 2000/Revised: 13 April 2000 相似文献
10.
The relationships between currents generated by the rabbit Na+/glucose cotransporter (SGLT1) and the fluxes of Na+ and sugar were investigated using Xenopus laevis oocytes expressing SGLT1. In individual voltage-clamped oocytes we measured: (i) the current evoked by 10 mmαMG and the 22Na+ uptake at 10 mm Na+; (ii) the currents evoked by 50 to 500 μm [14C]αMG and the [14C]αMG uptakes at 100 mm Na+; and (iii) phlorizin-sensitive leak currents in the absence of sugar and 22Na+ uptakes at 10 mm Na+. We demonstrate that the SGLT1 leak currents are Na+ currents, and that the sugar-evoked currents are directly proportional to both αMG and Na+ uptakes. The Na+/αMG coupling coefficients were estimated to be 1.6 at −70 mV and 1.9 at −110 mV. This suggests that the rabbit SGLT1 Na+/αMG stoichiometry for sugar uptake is 2 under fully saturating, zero-trans conditions. Coupling coefficients of less than 2 are expected under nonsaturating conditions due to uncoupled Na+ fluxes (slippage). The similarity between the Na+ Hill coefficients and the coupling coefficients suggests strong cooperativity between the two Na+ binding sites.
Received: 6 October 1997/Revised: 5 December 1997 相似文献
11.
Paula D. Raposinho Catarina Xavier João D. G. Correia Soraia Falcão Paula Gomes Isabel Santos 《Journal of biological inorganic chemistry》2008,13(3):449-459
Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several
linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin
type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly
enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH2]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage
of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization
on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the 99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH2 with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH2 in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing
a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with
the fac-[99mTc(CO)3]+ moiety. The 99mTc(CO)3-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic 99mTc(CO)3-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity,
after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells.
A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83
and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear 99mTc(CO)3-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the
potent (Nle4,dPhe7)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h
after injection for cyclic and linear radioconjugates, respectively). 相似文献
12.
The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR
mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion
followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting
search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass
numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags
search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly
identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination
reaction of l-cystathionine by the enzymological research. The K
m
value for the catalysis of l-cystathionine was 0.81 mM and V
max was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH3SeH by its α,γ-elimination reaction. 相似文献
13.
A method for regenerating pigeonpea [Cajanus cajan (L.) Millsp.] plants has been developed using distal cotyledonary segments of mature seeds as explants. A large number of
shoot buds were induced directly from explants of genotypes T-15-15 and GAUT-82-90 when cultured on six different basal media
fortified with 22.2 μm N6-benzylaminopurine, 2.3 μm kinetin, and 271 μm adenine sulfate. The shoot buds developed into shoots when they were subcultured on the same medium but with one-tenth concentrations
of cytokinins and adenine sulfate. The shoots elongated by subculturing first two to three times on Murashige and Skoog (MS)
basal medium supplemented with 2.22 μm N6-benzylaminopurine and 0.54 μm
α-naphthaleneacetic acid or on half-strength MS medium containing 2.89 μm gibberellic acid, and then once on the same medium without growth regulators. Elongated shoots were rooted with 80–85% efficiency
on MS medium with 4.92 μm indole-3-butyric acid and the plantlets were transferred for hardening. Plants survival in pots was 70–75%. This method may
be useful for improving the crop through genetic manipulations.
Received: 11 August 1997 / Revision received: 12 January 1998 / Accepted: 30 January 1998 相似文献
14.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
15.
Summary A thermostable NADP-dependent isocitrite dehydrogenase (IDH; EC. 1.1.1.42) was purified from the obligately thermophilic hydrocarbonoclastic bacterium Thermoleophilum minutum YS-4 (ATCC 35265). This was accomplished by affinity chromatography and electroelution from a nondenaturing polyacrylamide gel. The enzyme has an M
r of 60 000 and is composed of two identical subunits of M
r 30 500. The amino acid composition has an Arg/Lys ratio of 4:1 and very high levels of glycine. Under nondenaturing conditions, the enzyme has a distinct difference in electrophoretic mobility relative to IDHs obtained from other genera including the genus Thermus. The secondary strcuture consists of 16% -helix, 20% -sheet, 25% -turn and 37% random coil as determined by circular dichroism spectroscopy. The optimum pH and temperature for activity were 7.2 and 75° C respectively and the apparent K
mvalues for DL-isocitrate adn NADP+ were 33 M, and 48 M, respectively. The enzyme requires divalent cations, such as Mn2+ or Mg2+ for activity. NAD+ cannot substitute for NADP+. Oxaloacetate plus glyoxylate exert considerable inhibition on IDH activity while other glycolytic and tricarboxylic acid cycle intermediates have a lesser effect. p-Chloromercuribenzoic acid was inhibitory to the IDH although isocitrate and Mn2+ offered some protection from this inactivation. The enzyme is thermostable, retaining 84% and 57% of initial activity after incubation for 1 h at 60° and 70° C, respectively. Isocitrate provided protection from thermal inactivation allowing the IDH to maintain 21% activity after 1 h at 80° C.
Offprint requests to: J. J. Perry 相似文献
16.
Somatic embryogenesis and in vitro rosmarinic acid accumulation in Salvia officinalis and S. fruticosa leaf callus cultures 总被引:1,自引:0,他引:1
The effect of explant age, plant growth regulators and culture conditions on somatic embryogenesis and rosmarinic acid production
from leaf explants of Salvia officinalis and S. fruticosa plants collected in Greece was investigated. Embryogenic callus with numerous spherical somatic embryos could be induced
on explants derived from both species and cultured for 3 weeks on a Murashige and Skoog (MS) medium supplemented with 1.8–18
μm 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (Kin) or 10.5–21 μm 1-naphthalenacetic acid and 6-benzyladenine. Only explants from young plants (with six to eight leaves) responded to the
culture treatments and, in general, low light intensities (50 μmol m–2 s–1) favoured callus formation and induction of somatic embryos. Somatic embryos were further developed on the same medium. Heart-
and torpedo-shaped embryos (1–2 mm long) were subcultured on a growth-regulator-free MS medium for maturation. Maximum rosmarinic
acid accumulation in S. officinalis and S. fruticosa callus cultured on 4.5 μm 2,4-D and 4.5 μm Kin was 25.9 and 29.0 g/l, respectively.
Received: 17 January 1997 / Revision received: 26 May 1997 / Accepted: 30 June 1997 相似文献
17.
Guennoun-Lehmann S Fonseca JE Horisberger JD Rakowski RF 《The Journal of membrane biology》2007,216(2-3):107-116
Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric
H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α2- and Na,K-ATPase β2-subunits; Bufo Na,K-ATPase α1- and Na,K-ATPase β2-subunits; and Bufo Na,K-ATPase β2-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring 86Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β2-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α1-subunit and Na,K-ATPase β1-subunit; rat Na,K-ATPase α2-subunit and Na,K-ATPase β2-subunit; and rat Na,K-ATPase β1- or Na,K-ATPase β2-subunit alone. Measurement of increases in 86Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα1/NKβ1 and NKα2/NKβ2. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα1/NKβ1 exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected
with rat NKβ1- or rat NKβ2-subunit alone. However, in HeLa cells expressing rat NKα2/NKβ2, outward current was observed after pump activation by 20 mm K+ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act
on Na,K-ATPase. 相似文献
18.
When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L−1 medium-chain-length polyhydroxyalkanoate (PHAMCL) and 0.57 g L−1 alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHAMCL, which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T
m and T
g values for the PHAMCL were 51.03°C and −41.21°C, respectively, by DSC. Its decomposition temperature was about 300°C. The elongation at break was
700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights
of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed
that the AO were consisted of β-d-mannuronic acid and/or α-l-guluronic acid, and the β-d-mannuronic acid and/or α-l-guluronic acid residues were partially acetylated at position C2 or C3. 相似文献
19.
Solubilization of enzymes in water-in-oil microemulsions and their rapid and efficient release through use of a pH-degradable surfactant 总被引:3,自引:0,他引:3
α-Chymotrypsin and lysozyme were solubilized in a water/O-[(2-tridecyl, 2-ethyl-1,3-dioxolan-4-yl)methoxy]–O′-methoxy poly(ethylene glycol) (CK-2,13 surfactant)/isooctane water-in-oil microemulsion solution at 1.5–2 and 10 g l−1 for 0.15 and 1.2 M CK-2,13, respectively. Upon contact with an equal volume of 0.1 M NaH2PO4/Na2HPO4 buffer, pH 5, a three-phase system (Winsor-III system) was formed, consisting of a surfactant-rich middle phase and aqueous
and isooctane-rich “excess” phases. Both enzymes were rapidly released into the aqueous excess phase, with 70% recovery of
each in 30 and 60 min for microemulsion solutions containing 0.15 and 1.2 M surfactant, respectively. The recovered enzymes retained >90% of their original specific activity. 相似文献
20.
Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial
glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p
Gly
) was 2.3 ± 0.23 × 10−6 cm sec−1 with MIP vs. 0.92 ± 0.086 × 10−6 cm sec−1 in control oocytes. The p
Gly
of MIP was independent of concentration from 5 × 10−5 to 5 × 10−2
m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mm Hg++, 0.2 mm DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This
could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based
on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase
(0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min−1 cell−1) without changing the binding of glycerol to the kinase (K
M
∼ 10 μm).
Received: 23 May 1997/Revised: 4 August 1997 相似文献