The fusion of biological membranes entails a drastic rearrangement of the lipid bilayer. New assays that distinguish fusion from lysis were developed to study an in vitro reconstitution of the yeast vacuolar fusion machinery. These assays revealed that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and by lysis.
SUMMARY Growth of cells in contact with an abiotic or biological surface profoundly affects cellular physiology. In the opportunistic human pathogen, Candida albicans, growth on a semi-solid matrix such as agar results in invasive filamentation, a process in which cells change their morphology to highly elongated filamentous hyphae that grow into the matrix. We hypothesized that a plasma membrane receptor-type protein would sense the presence of matrix and activate a signal transduction cascade, thus promoting invasive filamentation. In this communication, we demonstrate that during growth in contact with a semi-solid surface, activation of a MAP kinase, Cek1p, is promoted, in part, by a plasma membrane protein termed Dfi1p and results in invasive filamentation. A C. albicans mutant lacking Dfi1p showed reduced virulence in a murine model of disseminated candidiasis. Dfi1p is a relatively small, integral membrane protein that localizes to the plasma membrane. Some Dfi1p molecules become cross-linked to the carbohydrate polymers of the cell wall. Thus, Dfi1p is capable of linking the cell wall to the plasma membrane and cytoplasm.
BackgroundContaminated hospital surfaces are an important source of nosocomial infections. A major obstacle in marketing antimicrobial surfaces is a lack of efficacy data based on standardized testing protocols.AimWe compared the efficacy of multiple testing protocols against several “antimicrobial” film surfaces.MethodsFour clinical isolates were used: one Escherichia coli, one Klebsiella pneumoniae, and two Staphylococcus aureus strains. Two industry methods (modified ISO 22196 and ASTM E2149), a “dried droplet”, and a “transfer” method were tested against two commercially available antimicrobial films, one film in development, an untreated control, and a positive (silver) control film. At 2 (only ISO) and 24 hours following inoculation, bacteria were collected from film surfaces and enumerated.ResultsCompared to untreated films in all protocols, there were no significant differences in recovery on either commercial brand at 2 or 24 hours after inoculation. The silver surface demonstrated significant microbicidal activity (mean loss 4.9 Log10 CFU/ml) in all methods and time points with the exception of 2 hours in the ISO protocol and the transfer method. Using our novel droplet method, no differences between placebo and active surfaces were detected. The surface in development demonstrated variable activity depending on method, organism, and time point. The ISO demonstrated minimal activity at 2 hours but significant activity at 24 hours (mean 4.5 Log10 CFU/ml difference versus placebo). The ASTEM protocol exhibited significant differences in recovery of staphylococci (mean 5 Log10 CFU/ml) but not Gram-negative isolates (10 fold decrease). Minimal activity was observed with this film in the transfer method.ConclusionsVarying results between protocols suggested that efficacy of antimicrobial surfaces cannot be easily and reproducibly compared. Clinical use should be considered and further development of representative methods is needed.
The genes rbcS and rbcL encode, respectively, the small and large subunits of the photosynthetic carbon dioxide fixation enzyme ribulose bisphosphate carboxylase͞oxygenase. There is a single rbcL gene in each chloroplast chromosome; a family of rbcS genes is located in the nuclear genome. These two genes are not ex- L eaves of C3 plants contain a single type of photosynthetic cell. Each of these cells carries on all of the reactions of oxygenic photosynthesis and contains the carbon dioxide-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase͞ oxygenase). This enzyme catalyzes the fixation of CO 2 to ribulose-1,5-phosphate and the production of two molecules of 3-phosphoglycerate.Maize is a C4 plant. Its leaves have two types of major photosynthetic cells: a cylinder of bundle sheath cells (BSC) surrounds each of the vascular bundles; mesophyll cells (MC) occupy the remainder of the space between the upper epidermis and the lower epidermis. Some MC and BSC are immediately adjacent to one another. In MC, CO 2 is fixed to phosphoenolpyruvate (PEP) by PEP carboxylase to form oxaloacetate, the latter, a four-carbon acid, is reduced to malate, which is transferred to BSC. CO 2 is released from the malate in BSC. Rubisco, which refixes the CO 2 , is present in BSC but not in MC. C4 species differ with regard to the exact product of oxaloactate that is moved from MC to BSC but in all cases Rubisco is found only in one cell type.Rubisco is comprised of eight large subunits, each of about 55 kDa, and eight small subunits, each of about 13 kDa. The large subunit is the product of a single chloroplast rbcL gene per chloroplast chromosome (1, 2). The small subunits are encoded by a family of nuclear rbcS genes (3).Transcripts of rbcS and rbcL are detectable in MC and BSC in leaves of dark-grown maize seedlings. Upon illumination the transcripts increase 2-to 3-fold in abundance in BSC but become undetectable in MC (3-5). The maize nuclear gene rbcS-m3 (6) follows the general pattern of rbcS expression in maize, i.e., expression is induced in BSC and repressed in MC upon illumination of dark-grown seedlings. About 35% of the total leaf rbcS mRNA in 24 h illuminated dark-grown maize is transcribed from rbcS-m3 (3). The control of repression of this gene in MC is the subject of the present work.Using an in situ reporter gene transient expression assay, we found previously that sequences of rbcS-m3 that lie between Ϫ93 bp and ϩ64 bp of the transcription start site are required for promoting photoregulated expression in BSC (7,8). On the other hand, photoregulated partial suppression of rbcS-m3-reporter gene expression in MC was found to require gene sequences that lie between Ϫ907 and Ϫ445 bp together with sequences that lie between ϩ720 bp and ϩ957 bp. The latter are just beyond the translation stop codon within the 3Ј transcribed region of the gene, but are equally effective when relocated 5Ј to the Ϫ907 to Ϫ445 corepressor-containing region. We also found that expression of the reporter gene is suppressed in MC only ...
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