A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts of Vicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. "Laser-assisted" patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.     

A versatile chamber for simultaneous measurements of oxygen exchange and fluorescence in filamentous and thallous algae as well as higher plants

A new chamber was developed for a simultaneous measurement of fluorescence kinetics and oxygen exchange in filamentous and thallous algae as well as in small leaves of water plants. Algal filaments or thalli are kept by a stainless grid close to the bottom window of the chamber in the sample compartment. The grid separates the object from the electrode compartment with the oxygen electrode at the top. This compartment accommodates, in addition, a magnetic stirrer that provides efficient circulation of the medium between the sample and the electrode. This magnetic bar spins on a fixed axis and is driven by an electronically commutated magnetic field produced by four coils which are arranged around the chamber. This design yields a very favourable signal to noise ratio in the oxygen electrode records. Consequently, measurements can be performed even of algae with very low photosynthetic rates such as marine low-light red algae or algae under severe stress. For irradiation of the samples and for fluorescence measurements a fibre optic light guide is used facing the window of the chamber. The four branches of a commercially available light guide serve the following purposes: collection of sample fluorescence and supply of measuring, actinic, and saturating light, respectively.     

Pharmacokinetic investigation of a 14C-labelled beta 3/alpha tetrapeptide in rats

The solid-phase synthesis and an ADME investigation with albino and pigmented male rats of the doubly 14C-labelled beta/alpha-tetrapeptide derivative Ac-beta3 hTyr-(D)Trp-beta3 hLys-beta3 hThr-lactone (3; Fig. 3) are described. After intravenous (i.v.) and peroral (p.o.) administration of the peptide, its concentration in blood and plasma, its tissue distribution, and the metabolism and the excretion of the peptide were analyzed over a period of up to 7 days post dose. The tetrapeptide in its ring opened form, 5, has a bioavailability of ca. 25%; radioactivity is distributed in the animals in an organ-specific way, and the compound appears to pass the blood-brain barrier to a very small extent, if at all (Tables 1-3 and Figs. 2-6). Excretion (37% renal, 44% fecal, including biliary) of the tetrapeptide 4 days after i.v. administration is almost complete, with only 4.3% remaining in the carcass; 4 days after p.o. administration 97% of the dose has been excreted in the feces. Radiochromatograms taken of plasma (0.5 and 24 h after i.v. dosing) and of urine and feces extracts (0-48 h collected) reveal the presence of lactone 3 and/or the corresponding hydroxy acid 5 with essentially no or very minor other peaks, respectively, representing possible metabolites (Tables 4-6, and Fig. 7 and 8). A comparison with a previous ADME investigation of a beta-nonapeptide show that--except for the lack of metabolism--all aspects of exposure, distribution, and elimination are different (structure-specific properties). The investigated tetrapeptide 3 is a potent and highly specific agonist of the somatostatin receptor hsst4, rendering the results described herein promising for diagnostic and therapeutic applications of beta-peptides.     

Maternal-fetal transplacental leakage of mitochondrial DNA in bovine nuclear transfer pregnancies: potential implications for offspring and recipients

The synepitheliochorial placenta of ruminants is constructed of multiple tissue layers that separate maternal and fetal blood. In nuclear transfer cloned ruminants, however, placental anomalies such as abnormal vascular development and hemorrhagic cotyledons have been reported. We have investigated the possible exchange of genetic material between somatic cell nuclear transfer cloned (SCNT) bovine fetuses and recipients at day 80 of gestation using mitochondrial DNA (mtDNA) as a marker. Twenty-three recovered SCNT-fetuses and their recipients were screened for divergent and thus informative mtDNA combinations. Twenty-one fetuses generated by in vitro fertilization (IVF) or multiple ovulation embryo transfer (MOET) and the corresponding recipients served as controls. A search for recipient mtDNA haplotype in DNA extracts from fetal blood by PCR-RFLP analysis revealed three cases of chimerism (two SCNT, one IVF) among a total of 19 informative fetus-recipient pairs (eight SCNT, seven IVF, four MOET). Placental anomalies have also been observed in some IVF fetuses and the present data therefore suggests transplacental leakage of cell components or cells from the recipient into some fetuses generated by in vitro techniques. Further studies are necessary to determine (i) the nature of leaked material, (ii) whether there is bi-directional leakage, and (iii) whether leaked material is present in recipients and calves after parturition, i.e. whether leakage takes place in vivo. If recipients were chimeric for DNA or cells derived from genetically modified SCNT (or IVF) embryos, their subsequent utilization might be affected.     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.     

Bacillus subtilis hydrolyzes CheY-P at the location of its action,the flagellar switch

In this report we show that in Bacillus subtilis the flagellar switch, which controls direction of flagellar rotation based on levels of the chemotaxis primary response regulator, CheY-P, also causes hydrolysis of CheY-P to form CheY and Pi. This task is performed in Escherichia coli by CheZ, which interestingly enough is primarily located at the receptors, not at the switch. In particular we have identified the phosphatase as FliY, which resembles E. coli switch protein FliN only in its C-terminal part, while an additional N-terminal domain is homologous to another switch protein FliM and to CheC, a protein found in the archaea and many bacteria but not in E. coli. Previous E. coli studies have localized the CheY-P binding site of the switch to FliM residues 6-15. These residues are almost identical to the residues 6-15 in both B. subtilis FliM and FliY. We were able to show that both of these proteins are capable of binding CheY-P in vitro. Deletion of this binding region in B. subtilis mutant fliM caused the same phenotype as a cheY mutant (clockwise flagellar rotation), whereas deletion of it in fliY caused the opposite. We showed that FliY increases the rate of CheY-P hydrolysis in vitro. Consequently, we imagine that the duration of enhanced CheY-P levels caused by activation of the CheA kinase upon attractant binding to receptors, is brief due both to adaptational processes and to phosphatase activity of FliY.