Subconductance states of single sodium channels modified by chloramine-T and sea anemone toxin in neuroblastoma cells

Single channel currents of chloramine-T (Chl-T) and sea anemone toxin (ATX-II) modified sodium channels were studied in neuroblastoma cells. With both substances similar subconductance states have been observed. The conductances of the sublevels were multiples of the unit step which was about onefourth of the most frequently occurring main conductance. Thus, the current levels observed were one fourth, half and five-fourths of the main current size. Both substances caused a slower decay of the averaged current compared to the current of the native channels. The main single-channel conductance was 15.2 pS (T=16°C) for the Chl-T and 10.8 pS (T=12°C) for the ATX-II modified channels. The channel open time was doubled by ATX-II, but was not increased significantly by Chl-T. The existence of the subconductance states suggests that the native channels may also have multiple open conformations.     

Effect of short-and long-term exposure to low environmental temperature on brain regional GABA metabolism

Single exposure of adult male rats to low environmental temperature (LET, 12 ± 0.5°C) for 2 h significantly increased the hypothalamic and striatal GABA levels without affecting those in other regions of brain. The activity of glutamate decarboxylase (GAD) was elevated in hypothalamus (H) and corpus striatum (CS) under these conditions. GABA accumulation rate (measured with ethanolamine-O-sulfate, an inhibitor of GABA-transaminase) was also increased in both H and CS of rats exposed to LET for 2 h. Unlike after a single exposure, the repeated exposure (2 h/day) for 7, 15, and 30 onsecutive days did not change the hypothalamic GABA metabolism. No change in GABA metabolism was observed in CS when rats were repeatedly exposed to LET for 7 consecutive days. Prolongation of repeated exposure to LET (2 h/day) for 15 and 30 consecutive days decreased the striatal GABA level and increased the activity of GABA-transaminase, although GAD activity was not altered under these conditions. These results suggest that single exposure to LET accelerates GABA synthesis and may reduce the GABAergic activity in both H and CS; whereas repeated exposure to LET for 15 or 30 consecutive days enhances GABAergic activity with the stimulation of GABA utilization only in CS without affecting its synthesizing process. Thus, it may be suggested that the hypothalamic and striatal GABA system may play a characteristic role in response to short-and long-term exposure to LET.     

Micropropagation of Plumbago rosea Linn.

Multiple shoot formation from the medicinal plant Plumbago rosea Linn. was induced on callus from stem segments on Murashige & Skoog media containing auxin and cytokinin. 2,4-D (2.5 mg l^-1) and kinetin (1.5 mg l^-1) added to the media gave best callus production, while BAP (2 mg l^-1) plus NAA (1.0 mg l^-1) induced shoot formation from that callus. Numerous shoots with roots could be produced by transferring shoots to media containing IBA (1.5 mg l^-1). Regenerated plantlets were transferred to pots and 60% survived.     

Kinetics of channelized membrane ions in magnetic fields

The cyclotron resonance model for channel ion transport in weak magnetic fields is extended to include damping losses. The conductivity tensor is obtained for different electric field configurations, including the circuital field E phi normal to the channel axis. The conductivity behavior close to the cyclotron resonance frequency omega c is compared to existing Ca2+-efflux data in the literature. A collision time of .023 s results from this comparison under the assumption that K+ ions are transiting in a 0.35 G field. We estimate a mean kinetic energy of 3.5 eV for this ion at resonance. This model leads to discrete modes of vibration (eigenfrequencies) in the ion-lattice interaction, such that omega n = n omega c. The presence of such harmonics is compatible with recent results by Blackman et al. [1985b] and McLeod et al. [1986] with the interesting exception that even modes do not appear in their observations, whereas the present model has no restriction on n. This harmonic formalism is also consistent with another reported phenomenon, that of quantized multiple conductances in single patch-clamped channels.     

Evidence for increased lipid peroxidation in multiple sclerosis

Pentane and ethane are degradation products of unsaturated fatty acids which are released during lipid peroxidation. In order to assess whether multiple sclerosis is associated with lipid peroxidation, we measured pentane and ethane excretion by 16 patients with multiple sclerosis and compared them to healthy control subjects. Patients with acute exacerbation of multiple sclerosis had significantly higher concentrations of pentane (10.5±4.2 nmol/l)(p<0.01) compared to either patients in remission (4.5±1.7 nmol/l) or control subjects (4.9±1.1 nmol/l). The concentrations of ethane were not significantly different among these groups. Of the patients with acute exacerbation who later achieved remission, the pentane excretion also returned to normal (5.6±0.9 nmol/l). One patient who failed to reachieve clinical remission continued to excrete large amounts of pentane. We conclude that oxygen free radical activity is enhanced during exacerbation multiple sclerosis.     

Multiplicity of soluble glucan-synthase activity in spinach leaves: Enzyme pattern and intracellular location

Buffer-extractable proteins from leaves of Spinacia oleracea L. were separated by non-denaturing polyacrylamide gel electrophoresis. Gels were stained for adenosine diphosphoglucose (ADPglucose)-dependent glucan-synthase (GS) activity (EC 2.4.1.21). Three major forms of activity were observed. No staining was detectable when ADPglucose was replaced by an equimolar concentration of either uridine, guanosine or cytosine diphosphoglucose. Two of the three GS forms exhibited both primed and citrate-stimulated unprimed activity whereas one enzyme form was strictly dependent upon the presence of an exogenous glucan. For intracellular localization, mesophyll protoplasts and intact chloroplasts were isolated and their enzyme pattern was compared with that of the leaf extract. Intactness and purity of the chloroplast preparations were ascertained by polarographic measurement of the ferricyanide- or CO₂-dependent oxygen evolution, by determination of marker-enzyme activities, and by electrophoretic evaluation of the content of chloroplast- and cytosol-specific glucanphosphorylase forms (EC 2.4.1.1). The three GS forms were present in mesophyll protoplasts. Intact chloroplasts possessed both primer-independent enzyme forms but lacked the primer-dependent one. The latter form was enriched in supernatant fractions of leaf homogenates when the intact chloroplasts had been pelleted by centrifugation. Thus, in spinach-leaf mesophyll cells soluble ADPglucose-dependent GS is located both inside and outside the chloroplast.Abbreviations GS glucan synthase - PAGE polyacrylamide gel electrophoresis This work has been made possible by grants from the Deutsche Forschungsgemeinschaft and from the Minister für Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The authors gratefully acknowledge the generous permission to use the laser densitometer of Professor Dr. W. Barz (Biochemie der Pflanzen, Universität Münster, FRG). They are indebted to Dr. H.-J. Witt (Pflanzenphysiologie, Universität Kassel, FRG) for helpful discussions and to Mr. W. Lamkemeyer for skilfull technical assistance.     

A map of barley chromosome 2 using isozymic and morphological markers

The F₂ progeny of a cross between a chromosome 2 multiple marker stock and an adapted cultivar of barley were analyzed for four morphological markers and electrophoretic patterns of eight leaf isozymes. TheIdh-2 locus was linked to thePer-5 locus (27.96±5.07 cM) and to thee locus (10.26±3.13 cM). Also, thePer-5 ande loci were located on the short arm of chromosome 2. In additionIdh-2 was also located on barley chromosome 2 and was linked to thev locus (13.18±3.56 cM), which is located on the long arm of chromosome 2. Two other marker genes,li andwst,,B, were linked (26.50±5.24 cM) on chromosome 2 but segregate independently of the other loci evaluated. This project was supported by funds from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.     

Versatile shuttle cloning vectors for the unicellular cyanobacterium Anacystis nidulans R2

Abstract 3 new shuttle cloning vectors for gene transfer into Escherichia coli and Anacystis nidulans have been constructed by utilizing the cyanobacterial origin of replication of the small plasmid pANS from A. nidulans . 2 of these new vectors, pXB7 (pDPL13 derivative) and pECAN8 (pUC8 derivative), convey ampicillin resistance, and transform A. nidulans with relatively high frequencies. Vector pXB7 has 10 unique cloning sites; pECAN8 contains 4 cloning sites within the lacZ gene permitting rapid detection of DNA inserts in the presence of Xgal. The third vector, pKBX, has a lower transformation frequency but adds kanamycin resistance as a selectable gene for shuttle vectors of cyanobacteria.