Water flow from soil to plants depends on the properties of the soil next to roots, the rhizosphere. Although several studies showed that the rhizosphere has different properties than the bulk soil, effects of the rhizosphere on root water uptake are commonly neglected. To investigate the rhizosphere's properties we used neutron radiography to image water content distributions in soil samples planted with lupins during drying and subsequent rewetting. During drying, the water content in the rhizosphere was 0.05 larger than in the bulk soil. Immediately after rewetting, the picture reversed and the rhizosphere remained markedly dry. During the following days the water content of the rhizosphere increased and after 60 h it exceeded that of the bulk soil. The rhizosphere's thickness was approximately 1.5 mm. Based on the observed dynamics, we derived the distinct, hysteretic and time-dependent water retention curve of the rhizosphere. Our hypothesis is that the rhizosphere's water retention curve was determined by mucilage exuded by roots. The rhizosphere properties reduce water depletion around roots and weaken the drop of water potential towards roots, therefore favoring water uptake under dry conditions, as demonstrated by means of analytical calculation of water flow to a single root.

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Resistance against ␤-lactam antibiotics is a growing challenge for managing severe bacterial infections. The rapid and costefficient determination of ␤-lactam resistance is an important prerequisite for the choice of an adequate antibiotic therapy. ␤-Lactam resistance is based mainly on the expression/overexpression of ␤-lactamases, which destroy the central ␤-lactam ring of these drugs by hydrolysis. Hydrolysis corresponds to a mass shift of ؉18 Da, which can be easily detected by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Therefore, a MALDI-TOF MS-based assay was set up to investigate different enterobacteria for resistance against different ␤-lactam antibiotics: ampicillin, piperacillin, cefotaxime, ceftazidime, ertapenem, imipenem, and meropenem. ␤-Lactamases are enzymes that have a high turnover rate. Therefore, hydrolysis can be detected by MALDI-TOF MS already after a few hours of incubation of the bacteria to be tested with the given antibiotic. The comparison of the MS-derived data with the data from the routine procedure revealed identical classification of the bacteria according to sensitivity and resistance. The MALDI-TOF MS-based assay delivers the results on the same day. The approved routine procedures require at least an additional overnight incubation.

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta‐cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP‐2 and cellubrevin, but not VAMP‐1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta‐cells contain secretory granules that store and secrete insulin as well as synaptic‐like microvesicles carrying gamma‐aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP‐2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP‐2 with insulin‐containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta‐cells. Pretreatment of streptolysin‐O permeabilized insulin‐secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP‐2 and cellubrevin and abolished Ca(2+)‐induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S‐stimulated insulin secretion. Taken together, our results show that pancreatic beta‐cells express VAMP‐2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)‐mediated insulin secretion.

Abstract. SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons.Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of ~ol and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca÷÷-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of '~60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and halfmaximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell typethe pancreatic B cell. IN pancreatic B cells, proinsulin is sorted in the transGolgi network for delivery to secretory granules where it is processed to insulin (23,40). Insulin is packed and stored in large dense core granules (LDCG) ~ and is released when exocytosis is stimulated by secretagogues such as glucose (23). Current understanding of B cell stimulussecretion coupling suggests that nutrient stimuli cause depolarization of the cell membrane, which leads to an influx of Ca ++ triggering the fusion of granules with the plasma membrane (48). Although sensitivity to glucose is a peculiarity of the pancreatic B cell, the other steps leading from the trans-Golgi network to LDCGs and the fusion of granules with the plasma membrane are, most probably, Please address all correspondence to Dr. Karin Sadoul, Laboratoires de Recherche Louis Jeantet, Centre Medical Universitaire, 1, rue MichelServet, CH-1211 Geneva 4, Switzerland. Tel.: 41 22 7025537. Fax.: 41 22 3473334. Abbreviations used in this paper:BoNT, botulinum neurotoxin; LDCG, large dense core granule; LDCV, large dense core vesicle; NSE N-ethylmaleimid-sensitive factor; SLMV, synaptic-like microvesicle; SNAP, soluble NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kD; SSV, small synaptic vesicle.common to all cells possessing the regulated secretory pathway.The molecular mechanism for docking and fusion of LDCGs in endocrine cells has not so far been studied. Conversely, recent data from S611ner et al. have established a detailed model for docking and fusion of small synaptic vesicles (SSV) in neuronal cells (57,58). Using cosedimentation and immunoprecipitation techniques they could identify a 20-S fusion complex. The complex is...

Depending on the size of the pores one wishes to produce in plasma membranes, the choice will probably fall on one of the three toxins discussed above. S. aureus alpha-toxin should be tried first when pores of 1-1.5 nm diameter are required. This is generally the case when Ca2+ and nucleotide dependence of a given process is being studied. If alpha-toxin does not work, this is probably due to the fact that the toxin either does not produce pores, or that the pores are too small. In this case, high concentrations of alpha-toxin should be tried. If this still does not work, we recommend the use of HlyA. When very large pores are to be created, e.g. for introduction of antibodies into the cells, SLO or another member of this toxin family are the agents of choice. SLO preparations need to be checked for presence of protease contaminants. Tetanolysin currently offers advantages since it is protease-free, and the size of the pores can probably be controlled by varying the toxin dose. Methods for assessing the size of pores created by such agents have been published in the recent literature, and the appropriate papers can be consulted whenever the need arises.

Pancreatic beta cells and cell lines were used in the present study to test the hypothesis that the molecular mechanisms controlling exocytosis from neuronal cells may be used by the beta cell to regulate insulin secretion. Using specific antisera raised against an array of synaptic proteins (SNAREs) implicated in the control of synaptic vesicle fusion and exocytosis, we have identified the expression of several SNAREs in the islet beta cell lines, beta TC6-f7 and HIT-T15, as well as in pancreatic islets. The v-SNARE vesicle-associated membrane protein (VAMP)-2 but not VAMP-1 immunoreactive proteins were detected in beta TC6-f7 and HIT-T15 cells and pancreatic islets. In these islet-derived cell lines, this 18-kDa protein comigrated with rat brain synaptic vesicle VAMP-2, which was cleaved by Tetanus toxin (TeTx). Immunofluorescence confocal microscopy and electron microscopy localized the VAMP-2 to the cytoplasmic side of insulin containing secretory granule membrane. In streptolysin O permeabilized HIT-T15 cells, TeTx inhibited Ca2+-evoked insulin release by 83 +/- 4.3%, which correlated well to the cleavage of VAMP-2. The beta cell lines were also shown to express a second vesicle (v)-SNARE, cellubrevin. The proposed neuronal target (t)-membrane SNAREs, SNAP-25, and syntaxin isoforms 1-4 were also detected by Western blotting. The beta cell 25-kDa SNAP-25 protein and syntaxin isoforms 1-3 were specifically cleaved by botulinum A and C toxins, respectively, as observed with the brain isoforms. These potential t-SNARES were localized by immunofluorescence microscopy primarily to the plasma membrane in beta cell lines as well as in islet beta cells. To determine the specific identity of the immunoreactive syntaxin-2 and -3 isoforms and to explore the possibility that these beta cells express the putative Ca2+-sensing molecule synaptotagmin III, RT-PCR was performed on the beta cell lines. These studies confirmed that betaTC6-F7 cells express syntaxin-2 isoforms, 2 and 2', but not 2'' and express syntaxin-3. They further demonstrate the expression of synaptotagmin III. DNA sequence analysis revealed that rat and mouse beta cell syntaxins 2, 2' and synaptotagmin III are highly conserved at the nucleotide and predicted amino acid levels (95-98%). The presence of VAMP-2, nSec/Munc-18, SNAP-25 and syntaxin family of proteins, along with synaptotagmin III in the islet cells and in beta cell lines provide evidence that neurons and beta cells share similar molecular mechanisms for Ca2+-regulated exocytosis. The inhibition of Ca2+-evoked insulin secretion by the proteolytic cleavage of HIT-T15 cell VAMP-2 supports the hypothesis that these proteins play an integral role in the control of insulin exocytosis.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75‐kbp plasmid from a toxigenic non‐sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.