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PubMed2001

Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains.

Synaptotagmin I (Syt I), a proposed major Ca(2+) sensor in the central nervous system, has been hypothesized as functioning in an oligomerized state during neurotransmitter release. We previously showed that Syts I, II, VII, and VIII form a stable SDS-resistant, beta-mercaptoethanol-insensitive, and Ca(2+)-independent oligomer surrounding the transmembrane domain (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the molecular mechanism of the Ca(2+)-independent oligomerization by the synaptotagmin family. In this study, we analyzed the Ca(2+)-independent oligomerization properties of Syt I and found that it shows two distinct forms of self-oligomerization activity: stable SDS-resistant self-oligomerization activity and relatively unstable SDS-sensitive self-oligomerization activity. The former was found to be mediated by a post-translationally modified (i.e. fatty-acylated) cysteine (Cys) cluster (Cys-74, Cys-75, Cys-77, Cys-79, and Cys-82) at the interface between the transmembrane and spacer domains of Syt I. We also show that the number of Cys residues at the interface between the transmembrane and spacer domains determines the SDS- resistant oligomerizing capacity of each synaptotagmin isoform: Syt II, which contains seven Cys residues, showed the strongest SDS-resistant oligomerizing activity in the synaptotagmin family, whereas Syt XII, which has no Cys residues, did not form any SDS-resistant oligomers. The latter SDS-sensitive self-oligomerization of Syt I is mediated by the spacer domain, because deletion of the whole spacer domain, including the Cys cluster, abolished it, whereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomerization of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys cluster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.

Amino Acid Sequence↗
PubMed1996

The generation of SDS-stable HLA DR dimers is independent of efficient peptide binding.

MHC class II molecules can exist in two conformations which can be distinguished on the basis of their stability in SDS. The formation of SDS-stable dimers has been shown to correlate with persistent expression of antigen MHC class II-peptide complexes by murine antigen-presenting cells. HLA DR molecules contain either a Val or a Gly at position 86 of the beta chain, which is located in a conserved and prominent hydrophobic pocket in the peptide binding site. Here we show that Val86-containing DR molecules more frequently select peptides which induce the formation of SDS-stable dimers than Gly86 variants. Using analogues of the influence virus haemagglutinin epitope 307-319 we found that the replacement of the aromatic hydrophobic anchor residue (Tyr)at position 309 by amino acids with an aliphatic hydrophobic side chain resulted in the specific formation of high numbers of SDS-stable Val86-DR but not Gly86-DR dimers. These results indicate that the fit between the first anchor residue and the hydrophobic pocket around Dr beta 86 plays a critical role in the formation of SDS-stable DR dimers. Synthetic analogues of naturally processed DR-associated peptides displayed promiscuity in their capacity to bind to several DR specificities and in their ability to induce the SDS-stable conformation. However, no correlation was observed between binding capacity and the ability to induce the SDS-stable conformation. Since it has been shown that SDS stability can relate to the kinetics of peptide-MHC class II interactions, the definition of the requirements for the formation of SDS-stable HLA class II molecules may be important for the design of effective peptide-based immunomodulation protocols.

Amino Acid Sequence↗
PubMed2003

Evaluation of SDS-polyacrylamide gel systems for the study of outer membrane protein profiles of clinical strains of Acinetobacter baumannii.

The outer membrane protein (OMP) profiles of 23 blood isolates of Acinetobacter baumannii representing all the different antimicrobial susceptibility patterns observed during a 3-year period in a Spanish hospital were studied. OMPs extracted from envelopes of sonicated cells after solubilisation with 2% of N-lauryl-sarcosinate were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using the Laemmli's buffers. Eight running gel systems differing in the concentration of polyacrylamide (8%, 10% and 12%) and in the absence or presence of urea (4 M and 6 M) were used in a preliminary study analysing the OMP profiles of four clonally unrelated strains of A. baumannii. When this study was completed, the OMPs of the 23 A. baumannii were analysed in 10% SDS-polyacrylamide gels with 6 M urea and in 12% SDS-polyacrylamide gels. Ten OMP profiles were observed in 10% SDS-polyacrylamide gels with 6 M urea, whereas only 5 OMP profiles were visualised using 12% SDS-polyacrylamide gels. The OMP profiles obtained in 10% SDS-polyacrylamide gels with 6 M urea only partially correlated with those observed in 12% SDS-polyacrylamide gels. In conclusion, the use of 10% SDS-polyacrylamide gels with 6 M urea is recommended for the study of OMP profiles of A. baumannii.

Acinetobacter Infections↗
PubMed1989

Variability in electrophoretic mobility of Gi-like proteins: effect of SDS.

Two forms of Gi-like protein are resolved in both somatic cells and mouse gametes when Sigma SDS (95% grade) is used during polyacrylamide gel electrophoresis, whereas only a single species is resolved when Bio-Rad SDS (electrophoresis grade) is used. These two Gi-like proteins are likely to reflect two distinct species, since (i) the two species resolved in the presence of Sigma SDS migrate with the same electrophoretic mobility upon re-electrophoresis in the presence of Sigma SDS and (ii) exchanging Sigma SDS for Bio-Rad SDS resolves a single species, whereas exchanging Bio-Rad SDS for Sigma SDS resolves two species.

Adenosine Diphosphate Ribose↗
PubMed2006

SDS (sodium dodecyl sulfate) effect on the autoxidation of the Glossoscolex paulistus giant extracellular hemoglobin: kinetic studies at pH 7.0 and 9.0.

The effect of the anionic surfactant sodium dodecyl sulfate (SDS) on the autoxidation process of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is addressed in the present work. The complex oligomeric assembly of hemoglobin subunits may influence the autoxidation rate and the exponential decay behavior. Kinetic studies were developed using UV-vis measurements at 415 nm. These spectroscopic measurements are analyzed at two pH values, 7.0 and 9.0, where the hemoglobin presents different oligomeric assembly. At pH 7.0 a high stability of the native form of the oxy-hemoglobin is observed, while at pH 9.0 an intense dissociation of the oligomer is promoted by alkalization. This difference is evident by comparison of the rate constants in the absence of surfactant: at pH 7.0 the kinetics presents a mono-exponential behavior with a rate constant of 0.27 x 10(-4)s(-1) while at pH 9.0 a bi-exponential behavior was observed with rate constant increase to 7 x 10(-4)s(-1) (fast process) and 1 x 10(-4)s(-1) (slow process). In the autoxidation induced by SDS two factors affect significantly the process rate, namely, the oligomeric arrangement of the hemoglobin and the strength of the interaction between SDS and HbGp. At pH 7.0, for SDS concentrations up to 0.3mM, a mono-exponential behavior was observed, showing rate constants around 0.4 x 10(-4)s(-1), which suggest that the hemoglobin still maintains the more compact structure observed at this pH for the native protein. In the SDS concentration range 0.75-1.0mM, the mono-exponential process changes into a bi-exponential behavior with rate constants varying from 48 x 10(-4) up to 99 x 10(-4)s(-1) for the fast process and from 1.7 x 10(-4) up to 3.7 x 10(-4)s(-1) for the slow process, suggesting hemoglobin dissociation. At pH 9.0, a bi-exponential decay is observed for all studied SDS concentration range, presenting rate constants from 11.0 x 10(-4) up to 179 x 10(-4)s(-1) for the fast process and from 1.0 x 10(-4) up to 8 x 10(4)s(-1) for the slow process probably due to hemoglobin dissociation, which is already present in the absence of surfactant. At pH 7.0, the highly packed native protein structure should inhibit the autoxidation process, but the SDS/HbGp interaction is more intense as compared to pH 9.0, due to the acid pI value, promoting oligomeric dissociation. So, the autoxidation process is regulated at pH 7.0 by the interaction with SDS, which triggers oligomeric dissociation and increase of autoxidation rate. At pH 9.0, the autoxidation process should be very fast, probably due to the oligomeric dissociation, which is already present in the absence of surfactant. At alkaline pH, the interaction with SDS seems be weaker than at pH 7.0. This behavior at pH 7.0 can be observed through the higher autoxidation rate for the faster chains and it is associated to the acid pI of the giant extracellular hemoglobins.

Animals↗
PubMed2006

Effects of SDS on the thermo- and pH-sensitive structural changes of the poly(acrylic acid)-based copolymer containing both poly(N-isopropylacrylamide) and monomethoxy poly(ethylene glycol) grafts in water.

The effects of SDS on the structural changes of the thermally induced polymeric micelles from a graft copolymer comprising poly(acrylic acid) (PAAc) as the backbone and poly(N-isopropylacrylamide) (PNIPAAm) and monomethoxy poly(ethylene glycol) (mPEG) as the grafts in aqueous solution are studied. At low temperature, SDS micelles form via the hydrophobic association of SDS molecules with the PNIPAAm grafts at a critical aggregation concentration of SDS (cac(SDS)) much lower than its critical micelle concentration. Consequently, the critical aggregation temperature of the graft copolymer is elevated. The corresponding structure of the thermally induced polymeric micelles is characterized by an abrupt reduction in the particle size and an increased tendency toward formation of the monocore structure with a more compact and hydrophobic PNIPAAm microdomain being developed. On the other hand, upon the polymeric micelle formation at high temperature, the copolymer-bound SDS micelle structure is disrupted and the dissociated SDS molecules migrate to the core-shell interface with their alkyl chains residing in the liquidlike region of the hydrophobic PNIPAAm microdomain. The correlation between the polymeric particles and copolymer-bound micelles is further substantiated by showing the change of the colloidal particle size in response to changes in cac(SDS) via adjusting the pH of the aqueous copolymer/SDS solutions.

Journal Article↗
PubMed1975

Proton magnetic resonance studies of the binding of an anionic surfactant with a benzene ring to a protein polypeptide with special reference to SDS-polyacrylamide gel electrophoresis.

The binding of an anionic surfactant to a protein polypeptide has been studied by the proton magnetic resonance (PMR) technique to form a part of our studies on the principles of SDS-polyacrylamide gel electrophoresis. Sodium 4-(p-butylphenyl) butane-1-sulfonate (CH3-(CH2)3-0-(CH2)4-SO3-Na+) was employed as an anionic surfactant, and reduced and carbosyamidomethylated (RCAM) bovine serum albumin as a typical protein polypeptide. The binding isotherm of the surfactant to RCAM bovine serum albumin was similar to that of sodium dodecyl sulfate (SDS). The surfactant could replace SDS in SDS-polyacrylamide gel electrophoresis without affecting the wellknown mode of spearation of protein bands. These results gave a sound basis for the assumption that the investigation of the complex between a surfactant with a benzene ring and RCAM bovine serum albumin would provide useful knowledge concerning the principles of SDS-polyacrylamide gel electrophoresis. Aggregation of the aromatic surfactant necessarily brings benzene rings together. A benzene ring is a strong source of the ring current effect on chemical shifts in nuclear magnetic resonance (NMR). Chemical shifts of the surfactant in NMR are, therefore, sensitive to whether the surfactant molecules are single-molecularly dissolved or aggregated. Full advantage was taken of the above fact in the present PMR study of the binding of the surfactant to RCAM bovine serum albumin. The chemical shifts of the phenyl and methyl protons both for the single-molecular and micellar aggregated states were estimated from measurements of the shifts as a function of the surfactant concentration. They shifted to a higher magnetic field on micelle formation, due to the increase of the ring current effect. Corresponding measurements for the complex between the surfactant and RCAM bovine serum albumin gave estimates of the chemical shifts of the phenyl and methyl groups of the surfactant bound to the protein polypeptide. They were found to shift to a magnetic field somewhat higher than that for the micellar state throughout the concentration range of the surfactant examined. These results strongly suggest that the surfactant molecules bind to the protein polypeptide in the form of micelle-like clusters, and that PMR of the groups are further influenced by the diagmagnetic effect of the protein polypeptide present as a core. No appreciable change in the mode of binding, corresponding to the steep increase in the amount of binding in the binding isotherm, was observed from the PMR studies. Taking the observed similarity between SDS and the aromatic surfactant in the binding and the gel electrophoresis into consideration, the present results strongly suggest that SDS also binds to protein polypeptides in the form of micelle-like clusters under the conditions of SDS-polyacrylamide gel electrophoreses, and support our "necklace model".

Benzene Derivatives↗
PubMed1994

SDS-degrading bacteria attach to riverine sediment in response to the surfactant or its primary biodegradation product dodecan-1-ol.

A laboratory-scale river microcosm was used to investigate the effect of the anionic surfactant sodium dodecyl sulphate (SDS) on the attachment of five Pseudomonas strains to natural river-sediment surfaces. Three of the Pseudomonas strains were chosen for their known ability to express alkylsulphatase enzymes capable of hydrolysing SDS, and the other two for their lack of such enzymes. One strain from each category was isolated from the indigenous bacterial population present in the river sediment used; other isolates were from soil or sewage. The alkylsulphatase phenotypes were confirmed by gel zymography of cell extracts. Addition of SDS to mixed suspensions of river sediment with any one of the biodegradation-competent strains stimulated the attachment of bacteria to the sediment particles. In contrast, the attachment of biodegradation-incompetent strains was weak and, moreover, was unaffected by SDS. The SDS-stimulated attachment for competent organisms coincided with rapid biodegradation of the surfactant. The primary intermediate of SDS biodegradation, dodecan-1-ol, accumulated transiently, and the numbers of attached bacteria correlated closely with the amount of dodecan-1-ol present. Direct addition of dodecan-1-ol also stimulated attachment but the effect was more immediate compared with SDS, when there was a lag period of approximately 2 h. To account for these observations, a model is proposed in which SDS stimulates the attachment of biodegradation-competent bacteria through its conversion to dodecan-1-ol, and it is hypothesized that the observed reversibility of the attachment is due to the subsequent removal of dodecan-1-ol by further bacterial metabolism.

Bacterial Adhesion↗
PubMed2002

Hybridization parameters revisited: solutions containing SDS.

Salt concentration governs nucleic acid hybridization according to the Schildkraut-Lifson equation. High concentrations of SDS are used in some common protocols, but the effects of SDS on hybridization stringency have not been reported. We investigated hybridization parameters in solutions containing SDS. With targets immobilized on nylon membranes and PCR- or transcription-generated probes, we report that the 50% dissociation temperature (Tm*) in the absence of SDS was 15 degrees C-17degrees C lower than the calculated Tm. SDS had only modest effects on Tm* [1% (w/v) equating to 8 mM NaCl]. RNA/DNA hybrids were approximately 11 degrees C more stable than DNA/DNA hybrids. Incomplete homology (69%) significantly reduced the Tm* for DNA/DNA hybrids (approximately /4degrees C; 0.45 degrees C/% nonhomology) but far less so for RNA/DNA hybrids (approximately 2.3 degrees C; approximately 0.07 degrees C/% non-homology); incomplete homology also markedly reduced the extent of hybridization. On these nylonfilters, SDS had a major effect on nonspecific binding. Buffers lacking SDS, or with low salt concentration, gave high hybridization backgrounds; buffers containing SDS, or high-salt buffers, gave reproducibly low backgrounds.

DNA Probes↗
PubMed2005

Comparative study of enzyme activity and stability of bovine and human plasmins in electrophoretic reagents, beta-mercaptoethanol, DTT, SDS, Triton X-100, and urea.

Effects of common electrophoretic reagents, reducing agents (beta-mercaptoethanol [BME] and DTT), denaturants (SDS and urea), and non-ionic detergent (Triton X-100), on the activity and stability of bovine plasmin (b-pln) and human plasmin (h-pln) were compared. In the presence of 0.1% SDS (w/v), all reagents completely inhibited two plns, whereas SDS (1%) and urea (1 M) denatured plns recovered their activities after removal of SDS by treatment of 2.5% Triton X-100 (v/v). However, reducing agents (0.1 M of BME and DTT) treated plns did not restore their activities. Based on a fibrin zymogram gel, five (from b-pln) and four (from h-pln) active fragments were resolved. Two plns exhibited unusual stability in concentrated SDS and Triton X-100 (final 10%) and urea (final 6 M) solutions. Two bands, heavy chain-2 (HC-2) and cleaved heavy chain-2 (CHC-2), of b-pln were completely inhibited in 0.5% SDS or 3 M urea, whereas no significant difference was found in h-pln. Interestingly, 50 kDa (cleaved heavy chain-1, CHC-1) of b-pln and two fragments, 26 kDa (light chain, LC) and 29 kDa (microplasmin, MP), of h-pln were increased by SDS in a concentration dependent manner. We also found that the inhibition of SDS against both plns was reversible.

Animals↗
PubMed2005

[A validation of the applicability of the Severity of Dependence Scale (SDS) in a nationwide mental hospital survey on substance-related psychiatric disorders].

We studied the applicability of the Severity of Dependence Scale (SDS) in a nationwide mental hospital survey on substance-related psychiatric disorders in 2002. The SDS is an easily administered five-item scale used to assess psychological dependence, especially the compulsive use of a wide range of substances. The SDS was translated into Japanese (SDS-J) and administered as a part of a questionnaire to 114 patients with substance-dependence syndrome in the survey. The SDS-J had a good internal consistency as demonstrated by the high Cronbach's alpha (0.76). Principal component analyses (PCA) with varimax rotation on the five items revealed a single-factor solution that accounted for 50.8% of the variance. Pearson's correlation coefficient between each item and total score ranged from 0.68 to 0.76, all of which were significant (p<.01). The total score of the SDS-J and the total number of items and psychological dependence-related items in the diagnostic guideline of dependence syndrome of the ICD-10 had rather low Pearson's correlation coefficients (0.17 and 0.23), but those correlations were significant (p < .05 and p < .01). The PCA for the patients with methamphetamine dependence also revealed a single-factor solution that accounted for 55.0% of the total variance. The results indicate that the SDS-J has satisfactory internal and structural reliability and external criteria validity. It also suggests that the SDS-J might be useful for assessing dependence syndrome in clinical settings in Japan, where those substances having properties of causing particularly severe psychological dependence, such as methamphetamine and solvents, are the most common substances of abuse.

Adult↗
PubMed1989

Protective immunity against Brugia malayi infective larvae in mice. II. Induction by a T cell-dependent antigen isolated by monoclonal antibody affinity chromatography and SDS-PAGE.

A mAb directed against filarial worm secretory/excretory product and reactive with Brugia malayi larval worm surface was used in conjunction with preparative SDS-PAGE to isolate protective Ag from extracts of adult B. malayi. The IgM mAb OVH bound to a repeating carbohydrate epitope present in adult, infective, and fourth stage larvae and microfilariae of B. malayi, and on the surface of fourth stage larvae. Ag bearing this epitope were also present in the sera of hosts infected with a variety of helminths, including Brugia, Onchocerca, Dirofilaria, and Paragonimus. Affinity chromatography of SDS extract of adult Brugia, using mAb OVH immobilized on agarose beads, isolated several Ag that separated into multiple protein staining bands on SDS-PAGE. In comparing SDS-PAGE-fractionated Ag from the crude SDS extract with fractionated mAb OVH-isolated Ag for the ability to protect BALB/c mice from challenge with B. malayi-infective larvae, it was found that of the mAb OVH-isolated Ag only those at a molecular mass of 26 to 32 kDa were protective while the original SDS extract yielded protective Ag at the following molecular mass: greater than 200, 170 to 200, 40 to 44, 33 to 36, 23 to 28, 20 to 22, and 17 to 19 kDa. Although Ag isolated by mAb OVH were highly protective, they failed to induce high antibody levels against the immunogen or SDS extracts compared to crude SDS extract immunized mouse sera, as determined by immunoblot and ELISA. Transfer of nylon wool non-adherent T cells from BALB/c mice immunized with the 26- to 28-kDa fraction of mAb OVH-isolated Ag to naive mice just before challenge with infective larvae of B. malayi resulted in a 70% reduction in larvae recovered 14 days after challenge.

Animals↗
PubMed1995

Sodium dodecyl sulfate (SDS) activation of the 20S proteasome in rat liver.

The peptidase activity of the 20S proteasome (multicatalytic protease complex) was examined in the 100,000g supernatant fraction prepared from rat liver tissue. Fluorogenic substrates for three proteasome peptidase activities were selected on the basis of (i) observation of an accelerated degradation in the presence of sodium dodecyl sulfate (SDS) and (ii) preferential degradation by the proteasome. Peptidase activities were assayed using an immunoprecipitation technique utilizing polyclonal antibodies raised against the purified rat proteasome. The ability to demonstrate SDS activation of the proteasome is shown to be dependent upon the choice of substrate. In addition, among the cytosolic peptidases, the property of SDS activation appears to be unique to the proteasome. SDS activation profiles were determined for each peptidase activity. Chymotrypsin-like and peptidylglutamyl peptide-hydrolyzing activities exhibit a broad plateau of activation between 0.04 and 0.05% SDS. Trypsin-like activity exhibits a sharp peak of activation at an SDS concentration of 0.04%. The SDS activation profile can be altered by changing the protein (proteasome) concentration, i.e., increasing protein (proteasome) concentration of the reaction mixture produces a marked rightward shift of the activation profile. On the other hand, changing the substrate concentration does not alter the profile. In conclusion, a technique for measuring proteasome peptidase activity in the 100,000g supernatant has been described. This approach increases the ease of measurement of peptidase activity and provides data which may more closely reflect the in vivo activity of the proteasome.

Amino Acid Sequence↗
PubMed1993

Selective removal of free dodecyl sulfate from 2-mercaptoethanol-SDS-solubilized proteins before KDS-protein precipitation.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the discontinuous system of Laemmli is used world-wide for analytical and preparative gel electrophoresis of polypeptides. A minor but disturbing problem is the difficulty of concentrating highly diluted solutions and determining their protein content after 2-mercaptoethanol-SDS solubilization. We describe a solution to both of these problems, detailing a two-step procedure which takes advantage of the low solubility of potassium dodecyl sulfate (KDS). Removal of excess of SDS and 2-mercaptoethanol, and concentration of proteins from even a nanomolar solution, is achieved by a two-step KDS precipitation. Free dodecyl sulfate is precipitated in step one, while KDS-proteins are pelleted in the second step, allowing the thiol agents to be discarded with the supernatant. The effects of changing [SDS] and [KC1], temperature, and pH were studied to optimize the separation of free SDS from proteins. After final precipitation, the hundred- or thousandfold concentrated proteins can be suspended in a small volume of any required medium. The procedure allows protein determination by the Lowry method, peptide mapping of 2-mercaptoethanol-SDS-solubilized polypeptides, and all other analyses which are otherwise hampered by excesses of SDS and/or thiol reagents.

Animals↗
PubMed1997

SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: practical procedure, interpretation and application.

Recently, we have developed a quick-freezing/freeze-fracture replica labeling technique, sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL), to study the two-dimensional distribution of cytochemical labeling on the membrane surface and the relationship of this distribution to images of freeze-fracture replicas created by platinum shadowing. In SDS-FRL, unfixed, quick-frozen cells, after freeze-fracture and platinum/carbon shadowing, are treated with SDS. The detergent dissolves unfractured areas of the cell membranes, with the release of the cytoplasmic contents. The cytoplasmic and exoplasmic membrane surfaces can be then labeled cytochemically. Integral membrane proteins, revealed as intramembrane particles by freeze-fracture replication, which are indistinguishable on a purely morphological basis, can be selectively labeled by SDS-FRL with specific antibody. In addition, this approach can be applied to examine the transmembrane phospholipid distribution in various cell and intracellular membranes. In this review, we describe the practical procedure for SDS-FRL in detail, present its application to labeling of various membrane components, and briefly discuss the possibility of a combination of SDS-FRL with atomic force microscopy.

Animals↗
PubMed1991

Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting.

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.

Animals↗
PubMed2006

Determining a cut-off on the Severity of Dependence Scale (SDS) for alcohol dependence.

Optimal cut-off points on the Severity of Dependence Scale (SDS) indicative of clinically significant dependence have been determined for a range of substance types. This study aims to determine a cut-off point on SDS that discriminates between the presence and absence of a DSM-IV diagnosis of alcohol dependence. A structured interview was administered to 90 alcohol users in Sydney, Australia. Receiver Operating Characteristic curve analysis confirmed the utility of the SDS-alcohol for characterising and diagnosing persons with respect to their alcohol-dependent status to an accuracy of 85%. A SDS score of 3 or above was determined as optimal for characterising alcohol dependence. Evidence is also provided confirming that the SDS-alcohol is a valid, reliable uni-dimensional scale for measuring alcohol dependence. It has been demonstrated that the SDS-alcohol can be used to characterise an individual's alcohol-dependent status. A cut-off value for SDS-alcohol provides additional meaning and value to the scale for clients and clinicians and will enable researchers to characterise the prevalence of alcohol dependence in their target populations.

Alcoholism↗
PubMed2005

The Severity of Dependence Scale (SDS) in an adolescent population of cannabis users: reliability, validity and diagnostic cut-off.

The Severity of Dependence Scale (SDS) is a five-item scale that has been reported to be a reliable and valid screening instrument for dependence and a measure of dependence severity in adults across several substance classes. To date no data have been reported on its performance in a population of adolescent cannabis users. The current study assessed the psychometric properties of the SDS in a community sample of 14-18-year-old adolescent cannabis users (n=100). Internal consistency (alpha=0.83) and test-retest coefficients (ICC=0.88) were high and a principal components analysis of the scale found all items to load on a single factor. Total SDS score correlated significantly with frequency of cannabis use and number of DSM-IV dependence criteria met, indicating good concurrent validity. Receiver Operating Characteristic curve analysis was used to determine the most appropriate SDS cut-off score for use as an indicator of cannabis dependence, with optimal discrimination at an SDS score of 4. These findings indicate that the SDS is a reliable and valid measure of severity of cannabis dependence among adolescents, has high diagnostic utility, and that an SDS score of 4 may be indicative of cannabis dependence.

Adolescent↗