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1.
M. Fucentese K.H. Winterhalter H. Murer J. Biber 《The Journal of membrane biology》1997,160(2):111-117
Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in
Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate
purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/P
i
-cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed
Na/P
i
-cotransport exhibited a K
m
of P
i
of 0.21 mm and an apparent K
m
of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected
with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher
compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a K
m
of SO2−
4 of about 0.3–0.4 mm and a K
m
of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaS
i
-1 proteins were detected at 55–60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters
for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered
by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing
conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography.
Received: 24 March 1997/Revised: 8 July 1997 相似文献
2.
R. Wondergem M. Cregan L. Strickler R. Miller J. Suttles 《The Journal of membrane biology》1998,161(3):257-262
These experiments were done to determine the effect of glibenclamide and diazoxide on the growth of human bladder carcinoma
(HTB-9) cells in vitro. Cell growth was assayed by cell counts, protein accumulation, and 3H-thymidine uptake. Glibenclamide added at 75 and 150 μm for 48 hr reduced cell proliferation. Dose-inhibition curves showed that glibenclamide added for 48 hr reduced cell growth
at concentrations as low as 1 μm (IC50= 73 μm) when growth was assayed in the absence of added serum. This μM-effect on cell growth was in agreement with the dose range
in which glibenclamide decreased open probability of membrane KATP channels. Addition of glibenclamide for 48 hr also altered the distribution of cells within stages of the cell cycle as determined
by flow cytometry using 10−5
m bromodeoxyuridine. Glibenclamide (100 μm) increased the percentage of cells in G0/G1 from 33.6% (vehicle control) to 38.3% (P < 0.05), and it reduced the percentage of cells in S phase from 38.3% to 30.6%. On the other hand, diazoxide, which opens
membrane KATP channels in HTB-9 cells, stimulated growth measured by protein accumulation, but it did not increase the cell number. We
conclude that the sulfonylurea receptor and the corresponding membrane KATP channel are involved in mechanisms controlling HTB-9 cell growth. However, KATP is not rate-limiting among the signaling mechanisms or molecular switches that regulate the cell cycle.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
3.
C. H. S. Carvalho N. Bohorova P. N. Bordallo L. L. Abreu F. H. Valicente W. Bressan E. Paiva 《Plant cell reports》1997,17(1):73-76
A total of 113 maize inbreds adapted to tropical conditions were evaluated for their tissue culture response. Additionally,
four media combinations of 15 or 30 μm dicamba with or without 88 μm AgNO3 were used to study the effect of dicamba and AgNO3 on type II callus production and plant regeneration from 42 of the inbred lines. Inbreds 48, 389 and 1345 of the populations
BR 105, BR 112, and Catete, respectively, showed a high capacity for type II callus production and plant regeneration. The
production of type II calli increased significantly when the concentration of dicamba was changed from 15 to 30 μm and when AgNO3 was added to the medium. A synergistic effect between 88 μm AgNO3 and 30 μm dicamba (CM-30Ag medium) was observed, leading to additional production of type II callus. Medium CM-30Ag allowed the best
tissue culture performance and plant regeneration capacity.
Received: 5 October 1996 / Revision received: 21 April 1997 / Accepted: 9 May 1997 相似文献
4.
The effects of pot size, timing of the application of paclobutrazol (PTZ) and gibberellic acid (GA3), and the counteractive effect of these two compounds on growth and tuber yield of greenhouse-grown Norland and Russet Burbank
potatoes were investigated. Plants were grown either in 1.5-liter pots (15 cm deep) or 3.0-liter pots (18 cm deep) and received
a foliar application of either 1.5 mm PTZ or 9 × 10−3 mm GA3 at early or late stolon initiation. Some plants that had been foliar treated with 1.5 mm PTZ at early stolon initiation were foliar treated with 9 × 10−3 mm GA3 at late stolon initiation. PTZ reduced haulm length in both cultivars significantly, particularly when the treatment was
applied at early stolon initiation, but the late treatment reduced haulm length only when growing in 3.0-liter pots. Irrespective
of the timing of treatment, GA3 increased haulm length in Norland growing in both pot sizes, but the treatment increased haulm length in Russet Burbank only
when applied at late stolon initiation. GA3 applied after PTZ did not overcome the growth-inhibiting effect of the PTZ treatment. The PTZ treatment effectively increased
usable tuber number/plant (UTN) in Norland, but PTZ had no effect on UTN in Russet Burbank. PTZ reduced usable tuber weight/plant
(UTW) only in Norland growing in 1.5-liter pots. By contrast, GA3 increased UTN only when treated at late stolon initiation of 1.5-liter pot-grown Norland, whereas the same treatment was
effective when applied only at early stolon initiation for Russet Burbank. For Norland, the increase in UTN by early applied
PTZ was reduced by the subsequent application of GA3. The use of 3.0-liter pots for minituber production in both Norland and Russet Burbank appears to have no advantage over
growing in 1.5-liter pots, particularly when PTZ or GA3 is used to enhance tuberization.
Received May 30, 1997; accepted February 3, 1998 相似文献
5.
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
6.
These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch
clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at −70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch
clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (KCa) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current
and the KCa activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V
m
) and 145 ± 13 pS at negative V
m
. Glibenclamide (50 μm), Ba2+ (1 mm) and quinine (100 μm) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50= 46 μm. The identity of this K+ channel with an ATP-sensitive K+ channel (KATP) was confirmed by its inhibition with ATP (2 mm) and by its activation with diazoxide (100 μm). We conclude that plasma membranes of HTB-9 cells contain the KCa and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked KATP.
Received: 12 June 1997/Revised: 21 October 1997 相似文献
7.
N.A. Sunstrom L.S. Premkumar A. Premkumar G. Ewart G.B. Cox P.W. Gage 《The Journal of membrane biology》1996,150(2):127-132
The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified
by affinity chromatography. NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions
with unequal concentrations (150–500 mm
cis, 50 mm
trans). An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2–3
mm), both depressed ion channel activity. Ion channels had a variable conductance, the lowest conductance observed being approximately
10 picosiemens. At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable
to sodium than to chloride ions (PNa/PCl∼ 9). In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium
potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa∼ 4). It was concluded that, at normal pHs, NB forms cation-selective channels.
Received: 6 March 1995/Revised: 17 November 1995 相似文献
8.
Skotodormant seeds of Lactuca sativa Grand Rapids imbibed in darkness for 10 days (10-day DS) germinated poorly upon terminal treatment with red light (R) or
gibberellin A3 (GA3). Soluble sugars in the imbibition solutions influenced the depth of skotodormancy. Ten-day DS seeds, imbibed in 50–500 mm sucrose or 100–500 mm glucose and given terminal GA3 germinated completely and germinated about 80% when imbibed in 100 mm galactose, mannose, lactose, or maltose. In contrast, terminal R applied to 10-day DS seeds caused only 20–50% germination.
If given R at day 0 and imbibed for 10 days in darkness in 500 mm sucrose or glucose, seeds washed free of exogenous glucose or sucrose then germinated about 50% in darkness in water. These
seeds responded to terminal R or GA3 with complete germination. When seeds were given FR at day 0, germination responses following terminal R or GA3 were significantly lower when the duration of DS was increased from 7–10 day DS to 15 days. In 10-day DS seeds given initial
FR and imbibed in either solutions of 50 or 100 mm sucrose and KNO3, either terminal R or GA3 treatment gave complete or near complete germination. It is concluded that seed exposure to certain soluble sugars and/or
nitrate during a 10-day DS protected certain substrates and thereby extended the sensitivity of the seeds to terminal R or
GA3 treatment. The study provides substantial evidence for nonhormonal factors associated with light and GA action in the control
of seed skotodormancy.
Received October 30, 1996; accepted April 22, 1997 相似文献
9.
Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system
The recently cloned Na/P
i
-cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P
i
-cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a K
m
for P
i
of 0.114 mm and an apparent K
m
for Na of 63 mm (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/P
i
-cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.We would like to thank W. Scherle and M. Lötscher (Institute of Anatomy) for their generous help using the confocal microscope and Ch. Gasser for the art work. Financial support by the Swiss National Fonds [Grant No. 32-30785.91 (to H.M.) and 32-28664.90 (to J.B.)] and by Stiftung für wissenschaftliche Forschung an der Universitát Zürich is greatly acknowledged. 相似文献
10.
Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula 总被引:1,自引:0,他引:1
Masahiro Nakao Masami Harada Yukiko Kodama Toru Nakayama Yuji Shibano Teruo Amachi 《Applied microbiology and biotechnology》1994,40(5):657-663
We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K
m=0.75 mm and molecular activity (k
cat)= 63.1 s–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K
m=0.04 mm
k
cat= 55.8 s–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl2) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl2). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose.
Correspondence to: T. Nakayama 相似文献
11.
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
12.
Haspel HC Stephenson KN Davies-Hill T El-Barbary A Lobo JF Croxen RL Mougrabi W Koehler-Stec EM Fenstermacher JD Simpson IA 《The Journal of membrane biology》1999,169(1):45-53
Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in
human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport
is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring
some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [3H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC50s of 0.2–2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic
side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all ≤10 mm) do not significantly inhibit glucose transport. The interaction of these three classes of anesthetics with purified GLUT-1
was evaluated by quenching of intrinsic protein fluorescence and displayed similar specificities and characteristics. The
ability of barbiturates to inhibit other facilitative glucose transporters was determined in cell types expressing predominantly
one isoform. Pentobarbital inhibits [3H]-2-deoxyglucose and [14C]-3-O-methyl-glucose uptake in cells expressing GLUT-1, GLUT-2, and GLUT-3 with IC50s of ∼1 mm. In contrast, GLUT-4 expressed in insulin-stimulated rat adipocytes was much less sensitive than the other isoforms to inhibition
by pentobarbital (IC50 of >10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms.
Received: 10 November 1998/Revised: 3 February 1999 相似文献
13.
We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40–50% of total Ca2+ uptake was inhibited by TG up to 10 nm (apparent Ki4.2 nm, Ca2+ pool I). An additional increase of inhibition up to 85–90% of total Ca2+ uptake could be achieved at 15 to 20 nm of TG (apparent Ki12.1 nm, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki10 m). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30–40% of total Ca2+ uptake was inhibited by 100 m of vanadate (apparent Ki18 m, Ca2+ pool II). The remaining 60–70% could be inhibited either by vanadate at concentrations up to 1 mm (apparent Ki300 m) or by TG up to 10 nm (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at 25% over a wide range of Ca2+ filling. About 10–20% remained unreleasable by IP3. Reduction of IP3 releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki 3.0 nm for IP3-induced Ca2+ release as compared to 4.2 nm for Ca2+ uptake at TG up to 10 nm) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations >10 nm which blocked Ca2+ pool II the apparent Ki values were 11.3 and 12.1 nm, respectively. For inhibition by vanadate up to 100 m the apparent Ki values were 18 m for Ca2+ uptake and 7 m for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mm the apparent Ki values were 300 and 200 m, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 m. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 m. In the presence of vanadate (inhibition of Ca2+ pool II), the apparent Km for IP3 was 2.1 m. These data allow the conclusion that there are at least three different Ca2+ uptake mechanisms present in pancreatic acinar cells: TG- and IP3 insensitive but highly vanadate-sensitive Ca2+ uptake occurs into membrane vesicles derived from plasma membranes. Two Ca2+ pools with different TG-, vanadate- and IP3-sensitivities are most likely located in the endoplasmic reticulum at different cell sites, which could have functional implications for hormonal stimulation of pancreatic acinar cells.This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 246. The authors wish to thank Dr. KlausDieter Preuß for valuable discussions and Mrs. Gabriele Mörschbächer for excellent secretarial help. 相似文献
14.
Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through
the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.660 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly.
However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium
solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation
reaction by the resting cells.
Received: 28 June 2001 / Accepted: 30 July 2001 相似文献
15.
F. Bigoni U. Koehler R. Stanyon T. Ishida J. Wienberg 《American journal of physical anthropology》1997,102(3):315-327
We employed in situ hybridization of chromosome-specific DNA probes (“chromosome painting”) of all human chromosomes to establish homologies between the human and the silvered lead monkey karyotypes (Presbytis cristata 2n=44). The 24 human paints gave 30 signals on the haploid female chromosome set and 34 signals on the haploid male chromosome set. This difference is due to a reciprocal translocation between the Y and an autosome homologous to human chromosome 5. This Y/autosome reciprocal translocation which is unique among catarrhine primates has produced a X1X2Y1Y2/X1X1X2X2 sex-chromosome system. Although most human syntenic groups have been maintained in the silvered leaf monkey chromosomes homologous to human chromosomes 14 and 15, 21 and 22 have experienced Robertsonian fusions. Further, the multiple FISH signals provided by libraries to human chromosomes 1/9, 6/16 indicate that these chromosomes have been split by reciprocal translocations. G-banding analysis shows three different forms of chromosome 1 (X2) which differ by a complex series of inversions in the 10 individuals karyotyped. Comparisons with the hybridization patterns in hylobatids (gibbons and siamang) demonstrate that resemblances in chromosomal morphology and banding previously taken to indicate a special phylogenetic relationship between gibbons and colobines are due to convergence. A. J. Phys. Anthropol. 102:315–327, 1997. © 1997 Wiley-Liss, Inc. 相似文献
16.
M.L. Tierney B. Birnir N.P. Pillai J.D. Clements S. M. Howitt G. B. Cox P.W. Gage 《The Journal of membrane biology》1996,154(1):11-21
The conserved leucine residues at the 9′ positions in the M2 segments of α1 (L264) and β1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant α1 subunits were co-expressed with normal or mutant β1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a
higher ``resting' chloride conductance than cells expressing wild-type α1β1 receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β1 with mutated α1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type
α1β1 receptors. In cells expressing mutated β1 subunits with wild-type or mutated α1 subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9′T mutation in the β1 subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an
open-channel state of the GABAA receptor in the absence of ligand.
Received: 17 April 1996/Revised: 5 July 1996 相似文献
17.
A K+ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid
bilayers. This channel was identified as an ATP-sensitive K+ channel (KATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na2. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0
mV and with a 5/250 KCl gradient, the main conductance of the KATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil
stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels.
In general, KATP channels were characterized by brief openings followed by long closings (open probability, P
o
≈ 0.02); only occasionally (3 out of 12 experiments) did the KATP channels have a high open probability (P
o
≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl2 to the internal side of the channel.
Received: 7 November 1995/Revised: 10 June 1996 相似文献
18.
Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mm NiCl2 in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mm NiCl2. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mm Ni2+) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5–5) isolated from nickel-rich soil in New Caledonia. 相似文献
19.
The uptake of 3H-labeled choline by a suspension of isolated type II epithelial cells from rat lung has been studied in a Ringer medium.
Uptake was linear for 4 min at both 0.1 μm and 5.0 μm medium choline; at 5 μm, only 10% of the label was recovered in a lipid fraction. Further experiments were conducted at the low concentration (0.1
μm), permitting characterization of the properties of high-affinity systems. Three fractions of choline uptake were detected:
(i) a sodium-dependent system that was totally inhibited by hemicholinium-3 (HC-3); (ii) a sodium-independent uptake, when
Na+ was replaced by Li+, K+ or Mg2+, inhibited by HC-3; (iii) a residual portion persisting in the absence of Na+ and unaffected by HC-3. Choline uptake was sigmoidally related to the medium Na+ concentration. Kinetic properties of the uptake of 0.1 μm
3H-choline in the presence and absence of medium Na+ were examined in two ways. (a) Inhibition by increasing concentrations of unlabeled choline (0.5–100 μm) was consistent with the presence of two Michaelis-Menten-type systems in the presence of Na+; a Na+-dependent portion (a mean of 0.52 of the total) had a K
m
for choline of 1.5 μm while K
m
in the absence of Na+ (Li+ substituting) was 18.6 μm. (b) Inhibition by HC-3 (0.3–300 μm) gave Ki values of 1.7 μm and 5.0 μm HC-3 for the Na+-dependent and -independent fractions. The apparent K
m
of the Na+-dependent uptake is lower than that reported previously for lung-derived cells and is in the range of the K
m
values reported for high-affinity, Na+-dependent choline uptake by neuronal cells.
Received: 18 February 1997/Revised: 7 December 1997 相似文献
20.
Both Pi-repressible acid phosphatases, IIb (mycelial) and IIc (extracellular), synthesized by Neurospora crassa and purified to apparent homogeneity by 7.5% PAGE, are monomers, are inhibited by 2 mm ZnCl2 and are non-specifically stimulated by salts. However, the IIc form is activated by p-nitrophenylphosphate (in a negative co-operativity effect with a K
0.5 of 2.5 mm) whereas form IIb shows Michaelis kinetics, with a K
m of 0.5 mm. Thus, since both enzymatic forms may be expressed by the same gene (pho-3), it is possible that post-translational modifications lead to the excretion of an enzymatic form with altered Michaelis kinetics compared with the enzymatic form retained by the mycelium. 相似文献