Biomedical subjects
Publications and source records attributed to M G Waters.
A simple sensor has been developed to detect the early stages of urinary catheter encrustation and avoid the clinical crises induced by catheter blockage. In laboratory models of colonization by Proteus mirabilis, the sensor signaled encrustation at an average time of 43 h before catheters were blocked with crystalline biofilm.
To identify novel factors required for ER to Golgi transport in yeast we performed a screen for genes that, when mutated, confer a dependence on a dominant mutant form of the ER to Golgi vesicle docking factor Sly1p, termed Sly1-20p. DSL1, a novel gene isolated in the screen, encodes an essential protein with a predicted molecular mass of 88 kDa. DSL1 is required for transport between the ER and the Golgi because strains bearing mutant alleles of this gene accumulate the pre-Golgi form of transported proteins at the restrictive temperature. Two strains bearing temperature-sensitive alleles of DSL1 display distinct phenotypes as observed by electron microscopy at the restrictive temperature; although both strains accumulate ER membrane, one also accumulates vesicles. Interestingly, the inviability of strains bearing several mutant alleles of DSL1 can be suppressed by expression of either Erv14p (a protein required for the movement of a specific protein from the ER to the Golgi), Sec21p (the gamma-subunit of the COPI coat protein complex coatomer), or Sly1-20p. Because the strongest suppressor is SEC21, we proposed that Dsl1p functions primarily in retrograde Golgi to ER traffic, although it is possible that Dsl1p functions in anterograde traffic as well, perhaps at the docking stage, as suggested by the suppression by SLY1-20.
STATEMENT OF PROBLEM: The wettability of silicone impression materials is poor, which may lead to voids within casts. All impression materials should be disinfected before use, but disinfection may affect their wettability. PURPOSE: This study evaluated the effect of disinfection procedures and the use of a surface wetting agent on the wettability of 4 addition-polymerized silicone impression materials. MATERIAL AND METHODS: Testing specimens were made from 4 addition silicone materials (light-bodied President, light-bodied Extrude, medium-bodied Extrude, and Aquasil). Two disinfection solutions (Actichlor and Perform) and 1 wetting agent (Vacufilm) were used. The test conditions were as follows: (A) dry, (B) Vacufilm, (C) Actichlor (10-minute soak), (D) Actichlor (10-minute soak) and Vacufilm, (E) Perform (10 minute-soak), and (F) Perform (10-minute soak) and Vacufilm. A dynamic contact angle analyzer was used to measure the wettability of specimens. Mean results were compared with 1-way ANOVA, and multiple comparisons were made with the Bonferroni method. RESULTS: Treatments C, D, and F had no significant effect on the wettability of the materials. Treatment B significantly reduced the contact angle for light-bodied President (P< .01) and Aquasil (P< .05). Treatment E significantly increased the contact angle for light- and medium-bodied Extrude and Aquasil (P< .001). CONCLUSION: Disinfection with Actichlor is recommended in preference to Perform to maintain the wettability of impression materials. Treatment with Vacufilm after disinfection is recommended to improve the wettability of materials and thus reduce the likelihood of voids within casts.
DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of approximately 80 and approximately 55 kDa. The approximately 80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is approximately 50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the delta-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction.
OBJECTIVE: To promote denture retention and denture comfort, denture materials should possess adequate wettability. This in vitro study investigated the wettability of nine commercially available dental materials. METHOD AND MATERIALS: Four denture base materials, two denture hard lining materials, and three denture soft lining materials (with and without varnish treatment) were tested. The wettability measurements were made using the dynamic contact angle analysis technique. The equilibrium and hysteresis angles obtained were used for the comparisons. RESULTS: The equilibrium contact angle (thetae) ranged from 63.9 (Permaflex + varnish) to 81.0 degrees (Mollosil + varnish). The differences observed among the materials tested were statistically significant. The contact angle hysteresis ranged from 16.0 (SR 3/60 Triplex) to 51.2 degrees (Mollosil), and there was a statistically significant difference among the materials. CONCLUSION: The heat-polymerized soft lining materials exhibited the greatest equilibrium contact angle, the autopolymerized soft liner had the lowest value, and the denture base materials had intermediate values. The soft liners showed greater contact angle hysteresis than all other materials. The use of varnish altered the wetting characteristics.
Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.
How do protein-transporting vesicles, which bud from the endoplasmic reticulum (ER), specifically dock to, and fuse with, the Golgi apparatus? In their Perspective, Brittle and Waters discuss new work (Allan et al.) suggesting that some vesicle-associated docking and fusion proteins are "programmed" during vesicle budding from the ER and direct downstream events that occur during fusion of these transport vesicles with the membranes of the Golgi.
Studies of intracellular trafficking over the past decade or so have led to striking advances in our understanding of the molecular processes by which transport intermediates dock and fuse. SNARE proteins play a central role, assembling into complexes that bridge membranes and may catalyze membrane fusion directly. In general, different SNARE proteins operate in different intracellular trafficking pathways, so recent reports that SNARE assembly in vitro is promiscuous have come as something of a surprise. We propose a model in which proper SNARE assembly is under kinetic control, orchestrated by members of the Sec1 protein family, small GTP-binding Rab proteins, and a diverse assortment of tethering proteins.
To evaluate the efficacy and safety of pentosan polysulfate sodium (PPS) in relieving symptoms of interstitial cystitis, the authors retrospectively reviewed charts of 260 patients in whom interstitial cystitis had been diagnosed. Subsequently, they conducted a follow-up phone interview or mail survey of those patients who were treated with PPS to investigate changes in the patients' symptoms, adverse effects, and change in quality of life. The control group consisted of patients whose interstitial cystitis had been diagnosed at cystoscopy and had a duration of at least 1 year and who had taken at least one or more oral medications for their symptoms. The average length of treatment was 9.3 months among the 27 subjects on PPS therapy. The mean length of time that they had diagnosed interstitial cystitis was 35.63 months and 48.78 months for the PPS-treated and control groups, respectively, with no statistically significant difference. Changes in frequency, urgency, and pain were greater in the treatment group and statistically significant (P = .11, P = .49, and P = .004, respectively). No change occurred in the rate of nocturia in the PPS-treated group compared with that in the control group. Symptoms of both groups improved over time, but improvement was statistically significantly greater in the treatment group (P = .001) over the treatment interval. The most common side effect attributable to PPS was diarrhea in 15% of subjects. Pentosan proved to be an efficacious option for reducing the debilitating symptoms of interstitial cystitis.
A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393-406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)-associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an approximately 750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.
STATEMENT OF PROBLEM: Maxillofacial prosthetic materials that contact skin or mucosa should have good wettability. A material that is easily wetted will form a superior lubricating layer between the supporting tissues and, thus, reduce friction and patient discomfort. The surface energy of a maxillofacial prosthetic material will give an indication of the amount of energy available for adhesion and of the susceptibility of the material to bacterial adhesion. PURPOSE: This study evaluated the wettability and surface energies of a range of commercially available silicone rubber maxillofacial prosthetic materials. MATERIAL AND METHODS: Contact angles and surface energies were measured by using a dynamic contact angle measuring technique. Four commonly used silicone maxillofacial materials were tested and their properties compared with those of an acrylic resin denture base material and a widely used denture soft lining material. RESULTS: There were no significant differences in the wettability of the silicone rubber materials. All materials were significantly less wetted than the denture acrylic resin material. There were no significant differences in the surface energies of the silicone rubber materials, but all were significantly lower than denture acrylic resin material. CONCLUSIONS: The Cahn dynamic contact angle analyzer was a quick and reproducible method for determining the contact angles and surface energies of maxillofacial materials. Further work is needed to improve the wettability of silicone rubber materials used for maxillofacial prostheses, thus, reducing their potential to produce friction with tissues.
OBJECTIVES: The objective of the present study was to evaluate the mechanical properties of an experimental formulation (Exp. 1) in order to assess its potential as a denture soft lining material. The same properties of a popular commercially available denture soft lining material (Molloplast-B) were determined and compared with the properties of Exp. 1. METHODS: Exp. 1 specimens were obtained by curing for 24 h at room temperature after the addition of the appropriate amounts of catalyst and cross-linker. Molloplast-B specimens were obtained after curing according to the manufacturer's instructions. The properties measured in the study were hardness, tear resistance, tensile strength and the bond strength of the material to a heat-cured acrylic denture base material. RESULTS: Exp. 1 had a significantly greater tensile strength, percent elongation, tear resistance and peel strength (p < 0.0001) than Molloplast-B. There was no significant difference in the hardness values of the two materials, although Molloplast-B had a significantly higher tensile bond and shear bond strength (p < 0.05). CONCLUSIONS: It was concluded that there was no significant difference in the hardness of Exp. 1 and Molloplast-B. Exp. 1 had superior tensile and tear properties. Its peel bond strength was superior to that of Molloplast-B, although its tensile bond strength and shear bond strength were less.
Studies of various membrane trafficking steps over the past year indicate that membranes are tethered together prior to the interaction of v-SNAREs and t-SNAREs across the membrane junction. The tethering proteins identified to date are quite large, being either fibrous proteins or multimeric protein complexes. The tethering factors employed at different steps are evolutionarily unrelated, yet their function seems to be closely tied to the more highly conserved Rab GTPases. Tethering factors may collaborate with Rabs and SNAREs to generate targeting specificity in the secretory pathway.
Intracellular protein traffic involves a tightly regulated series of events in which a membrane-bounded vesicles bud from one compartment and are specifically targeted to the next compartment, where they dock and fuse. A cell-free system that reconstitutes vesicle trafficking between the cis and medial Golgi cisternae has been used previously to identify several proteins involved in vesicular transport (N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment proteins, p115, and p16); however, these factors are insufficient to drive the transport reaction. We have used a modified version of this in vitro intra-Golgi transport assay to guide purification of a new transport-stimulating activity. The active component is a 13 S hetero-oligomeric complex consisting of at least five polypeptides (approximately 110, 109, 90, 82, and 71 kDa), which we term Golgi transport complex (GTC). Hydrodynamic properties suggest that GTC is approximately 800 kDa and nonglobular. We obtained peptide sequence information from the 90-kDa subunit (GTC-90) that allowed us to identify a number of GTC-90 cDNAs. Comparison of these cDNAs with one another and with the genomic sequence suggests that the GTC-90 mRNA is alternatively spliced. Anti-GTC-90 antibodies inhibit the in vitro Golgi transport assay, confirming the functionality of the purified complex. Subcellular fractionation indicates that GTC-90 exists in both membrane and cytosolic pools, with the cytosolic pool associated exclusively with the GTC complex. The membrane-associated pool of GTC-90 is localized to the Golgi apparatus.
Using a cell-free assay designed to reconstitute cis-to-medial intra-Golgi vesicular transport, we identified at least four crude activities in bovine brain cytosol that stimulate this assay. We have purified one of these activities to near homogeneity and have identified this Mr 36 kDa protein to be the alpha isoform of phosphatidylinositol transfer protein (PITPalpha) by N-terminal peptide sequencing, immunoreactivity with PITP-specific antisera, and the ability of recombinant PITPalpha to stimulate in vitro intra-Golgi transport. From these data, we conclude that in vitro Golgi transport is facilitated by PITPalpha.
SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (. Genetics. 142:393-406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target -SNARE-associated protein Sly1p. Weaker suppression is evident upon overexpression of genes encoding the vesicle-SNAREs SEC22, BET1, or YKT6. The cold-sensitive lethality that results from deleting SEC35 is suppressed by YPT1 or SLY1-20. These genetic relationships suggest that Sec35p acts upstream of, or in conjunction with, Ypt1p and Sly1p as was previously found for Uso1p. Using a cell-free assay that measures distinct steps in vesicle transport from the ER to the Golgi, we find Sec35p is required for a vesicle docking stage catalyzed by Uso1p. These genetic and biochemical results suggest Sec35p acts with Uso1p to dock ER-derived vesicles to the Golgi complex.
A novel approach for the assessment of adherence of Candida albicans to translucent acrylic material is described. The method uses the inverted microscope to visualise yeast adhering to acrylic surfaces while the test material remains immersed in buffer. Adherent cells were not subjected to surface tension forces that can occur during drying processes, so that an even distribution of yeast with no aggregation occurred. The process of counting attached yeast was subsequently performed without difficulty. From the 11 C albicans isolates examined, two groups were evident with respect to acrylic adherence: one group of four isolates with an adherence level of 400 yeast/mm2 acrylic, and one group of seven isolates with adherence levels of 1000 yeast/mm2 acrylic.
Intracellular vesicle targeting involves the interaction of vesicle proteins, termed v-SNAREs, with target membrane proteins, termed t-SNAREs. Assembly of v-SNARE-t-SNARE targeting complexes is modulated by members of the Sec1-Sly1 protein family, and by small guanosine triphosphatases termed Rabs. The interactions of these proteins during assembly of the endoplasmic reticulum-to-Golgi targeting complex in Saccharomyces cerevisiae were studied. The data suggest that the Rab protein Ypt1p transiently interacts with the t-SNARE Sed5p and results in displacement of the negative regulator Sly1p, allowing subsequent formation of the v-SNARE-t-SNARE targeting complex.