RESUMEN

The Centre for Data Linkage (CDL) was established at Curtin University, Western Australia, to develop infrastructure to enable cross-jurisdictional record linkage in Australia. The CDL's operating model makes use of the 'separation principle', with content data typically provided to researchers directly by the data custodian; jurisdictional linkage where available are used within the linkage process. Along with conducting record linkage, the team has also invested in establishing a research programme in record linkage methodology and in developing modern record linkage software which can handle the size and complexity of today's workloads. The Centre has been instrumental in the development of practical methods for privacy-preserving record linkage, with this methodology now regularly used for real-world linkages. While the promise of a nation-wide linkage system in Australia has yet to be met, distributed models provide a potential solution.

RESUMEN

The determination of cadmium levels in tissues of the tunicate Pyura stolonifera collected from uncontaminated sites revealed highest concentrations in the liver. After keeping P. stolonifera under laboratory conditions in Cd-containing water for 15 days, cadmium accumulated most markedly in liver tissue. Liver tissue of Cd-exposed specimens was used for the isolation of Cd-binding proteins. Five Cd-binding proteins, which differ in their chromatographic properties, could be purified. At least four of these Cd-binding proteins are heat stable and cysteine-rich. The N-terminal sequence (Met-Asp-Pro-Cys-Asn-Cys-Ala-Glu...) of at least two of these peptides resembles fish (plaice) metallothionein. Unlike vertebrate metallothioneins, P. stolonifera Cd-binding proteins are not N-terminally blocked by acetylation of methionine.

Asunto(s)

RESUMEN

A gene coding for Erythrina trypsin inhibitor (ETI) was designed, based on the published N-terminal sequence of the protein, and synthesized by an oligonucleotide-directed single strand break-repair mechanism. Direct expression from the expression vector pBtac1 was unsuccessful. A construct, encoding an extended methionyl N-terminal amino acid was expressed from the vector pET12a which supplies a signal sequence for export to the periplasm. Most of the expressed protein was located in the cytoplasm but because the periplasm is an environment conducive to the formation of disulphide bridges, only periplasmic protein was extracted. Cyanogen bromide cleavage at the sole methionyl residue removed the undesired amino acid residues that remained after signal sequence peptidase processing. The resultant ETI was assayed against trypsin and tissue plasminogen activator and found to have activity similar to that of natural ETI.

Asunto(s)

RESUMEN

Erythrina trypsin inhibitor (ETI) has good structural and sequence homology with soybean trypsin inhibitor (STI). However, STI does not inhibit tPA. From the three-dimensional structure of ETI it was known that the N-terminus of the molecule forms a finger-like structure stabilized by hydrogen bonds and hydrophobic interactions. In addition, the N-terminal finger region is located in close proximity to the reactive site loop and the N-terminal residue (Val) is bound up in the finger region. In STI the N-terminal region is located in close proximity to the reactive site loop and is folded into a structure similar to that in ETI. It was hypothesized that the N-terminal region is stabilized as in ETI and that the N-terminal residue of STI (Asp), because of its hydrophilic nature, is not involved in the structured N-terminal finger region of this protein. This leaves Asp1 of STI free to form an ion pair with Lys60 of trypsin, when STI and trypsin interact. When amino acid sequences of trypsin and the C-terminus of tPA are aligned for optimum homology, it is seen that there are a number of insertion sequences in tPA that are thought to be accommodated in the form of protrusions. One of these can be seen to occur in the region that lies opposite the Lys60 region of trypsin. It is suggested in this work that the N-terminal Asp of STI and this protrusion of tPA sterically prevent the two proteins from approaching close enough for binding and inhibition to occur. A modified form of ETI was produced with an Asp residue N-terminal to Val to simulate the N-terminal region of STI. The active sites were titrated against trypsin and assayed against tPA. The results showed that the modified form of ETI had activity towards tPA similar to that of STI. This evidence indicates strongly that the N-terminal Asp of STI prevents its binding to and inhibiting tPA.

Asunto(s)

RESUMEN

The value of magnetic resonance imaging (MRI) in the early diagnosis of avascular necrosis (AVN) of the hip in SLE was investigated. Twenty females with severe SLE were studied prospectively. Each underwent 6-monthly X-rays, technetium -99m (Tc-99m) pyrophosphate bone scans and MRI of the hips over a 3-yr period. AVN was diagnosed in five hips of three patients (15%) during the study period. It was confirmed histologically in three hips of two patients who underwent core decompression. Radiological evidence of AVN was present in two patients at diagnosis. One patient developed progressive radiological changes despite core decompression. Bone scintigraphy was abnormal at some stage in all three patients with AVN however failed to detect the early ischaemic stage of AVN. MRI was the most reliable investigation and was able to detect asymptomatic AVN prior to the appearance of radiological or scintigraphic abnormalities.

Asunto(s)

RESUMEN

We have designed and synthesized by conventional chemical techniques a 38mer oligonucleotide consisting of a 5'd(Pu)10d(C)4d(Py)10d(T)4d(Py)10(3') sequence. This oligonucleotide assumes a randomly coiled conformation at pH 12. At pH 8.0 a hairpin helix forms between its 5' purine decamer sequence and the consecutive pyrimidine decamer leaving the second pyrimidine decamer as a dangling disordered 3' extension. On reducing the pH to 4.5 this second pyrimidine decamer folds back onto the major groove of the hairpin helix resulting in an intramolecular triple-stranded stem-loop structure. We have used a variety of biochemical (gel mobility, P1 nuclease digestion) and biophysical (ultraviolet light and circular dichroism spectroscopy, fluorimetry, microcalorimetry) techniques to characterize the different conformers, their stability and the folding pathway into an intramolecular triple helix. The thermodynamic properties of this intramolecular triple strand in 100 mM-Na+ are: tm, 71 degrees C; delta HvH, 119.4(+/- 11.9) kcal mol-1; delta Hcal, 121.9 (+/- 6.1) kcal mol-1 at pH 4.5; those of the hairpin are: tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.9(+/- 3.5) kcal mol-1 at pH 8.0. At intermediate pH values, the triplex to coil transition breaks up into its component triplex to hairpin and hairpin to coil transitions with thermodynamic properties: tm, 41 degrees C; delta HvH, 58.7(+/- 4.2) kcal mol-1; delta Hcal, 39.8(+/- 2.0) kcal mol-1; and tm, 63 degrees C; delta HvH, 71.7(+/- 4.0) kcal mol-1; delta Hcal, 69.6(+/- 3.5) kcal mol-1 at pH 6.7.

Asunto(s)

RESUMEN

A cyclic tridecapeptide based on the sequence of an anti-tryptic loop of a Bowman-Birk inhibitor was synthesized, and demonstrated to be active as an inhibitor of trypsin. Molecular modeling of this sequence suggested an improved sequence which demonstrated an order of magnitude improvement in the inhibitory constant.

Asunto(s)

RESUMEN

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

Asunto(s)

RESUMEN

The toxin cyanoginosin-LA (MW 909), isolated from Microcystis aeruginosa, was successfully conjugated with polylysine and muramyl dipeptide to form a high molecular weight complex consisting of a hapten, a carrier and a built-in adjuvant. This complex was used for the immunization of mice. Monoclonal antibodies specific for cyanoginosin-LA were produced using the hybridoma technique. The ten most efficient producers of these antibodies were further characterized and the monoclonal antibody produced by them was found to be identical on the basis of additivity test, isoelectric focussing and sub-class immunoglobulin typing results. The anti-cyanoginosin-LA monoclonal antibody focussed at a pH of approximately 6.55 and was found to belong to the mouse immunoglobulin subclass IgM. Large scale production of the antibody (in vivo) was followed by purification with ammonium sulphate precipitation and high performance liquid chromatography. The anticyanoginosin-LA monoclonal antibody, when reacted with six variants of cyanoginosin (other than cyanoginosin-LA), bound all with equal efficiency.

Asunto(s)

RESUMEN

Monoclonal antibodies against Naja nivea cardiotoxin VII1 were produced using the hybridoma technique. The antibodies of two clones were found to be identical by an avidity test, isoelectric focusing and immunodiffusion typing assay. The monoclonal antibody was focused at a pH range of 7.4-8.1 and belonged to the mouse sub-class IgG1. A dissociation constant of 0.26 nM demonstrated its high affinity to cardiotoxin. The monoclonal antibody had no effect on cardiotoxin lethality or lysis of red blood cells by the toxin and could therefore be assumed to bind to an antigenic site separate from the active centre.

Asunto(s)

RESUMEN

Two phospholipases A2, CM-I and CM-II, were purified from Bitis nasicornis venom by gel filtration on Sephadex G-50, followed by ion-exchange chromatography on CM-cellulose. Both enzymes comprise 119 amino acids, including 12 half-cystine residues. The primary structure of CM-II has been elucidated. The sequence and invariant amino-acid residues of CM-II resemble those of phospholipases A2 from other venoms of Viperidae and Crotalidae (Group II) snake venoms. CM-I and CM-II both contain a single histidine residue which is probably located at the active centre (histidine-47). CM-II are relatively non-toxic.

Asunto(s)

RESUMEN

A presynaptic acting toxic phospholipase A2, designated caudoxin, was purified from the venom of Bitis caudalis by a combination of gel filtration and ion-exchange chromatography. The specificity of the enzyme was shown to be of the A2 type. The enzyme contains 121 amino acid residues in a single chain and is cross-linked by seven disulfide bridges. Application of cyanogen bromide cleavage and digestion with trypsin and chymotrypsin yielded peptides providing the necessary overlaps to complete derivation of the sequence. Structural features of caudoxin in relation to other toxic and non-toxic phospholipases A2 are discussed.

Asunto(s)

RESUMEN

Caudoxin, a single-chain phospholipase A2 isolated from the venom of Bitis caudalis is a toxic polypeptide with a formula weight of 13,332 dalton. The LD50 in mice (i.p.) was 0.18 (0.15-0.22) mg/kg. In the chick biventer cervicis muscle preparation the toxin (1-10 micrograms per ml) caused complete neuromuscular blockade without affecting the response of the muscle to acetylcholine. In the mouse phrenic nerve-diaphragm preparation, the toxin abolished the indirectly elicited contraction without inhibiting that evoked directly. When this preparation was bathed in a low calcium (0.6 mM) medium, the toxin induced a triphasic change in the indirectly evoked contractions: an immediate initial inhibition followed by augmentation and then a second phase of inhibition leading to irreversible neuromuscular blockade. Electrophysiological studies in the same preparation showed a similar triphasic change in the quantal content of endplate potentials. The frequency of miniature endplate potentials first increased and then decreased, while the resting membrane potential was not significantly decreased by the toxin. Histological study showed that the toxin caused local myonecrosis only at a higher dose (2 mg/kg mouse). It is concluded that caudoxin produced a neuromuscular block by acting selectively on a presynaptic site. However, the site of binding appears to be different from that of beta-bungarotoxin since combination of the toxin with beta-bungarotoxin caused potentiation of its neuromuscular blocking action rather than addition.

Asunto(s)

RESUMEN

The configuration assignment of the alpha-carbon atom of amino acid residues in four toxin variants from Microcystis aeruginosa have been made by stereospecific enzymic transformations. The relative conformation assignment of the beta-carbon atom of beta-CH3-aspartic acid could be made by comparison of the electrophoretic mobility with literature values reported for the authentic compound. The presence of an N-methyldehydroalanine residue, which, due to elimination of methylamine under hydrolytic conditions, previously escaped detection by conventional means, has been confirmed by identification of N-methylalanine in the hydrolysate after reduction of toxin with sodium borohydride.

Asunto(s)

RESUMEN

Two alternative procedures for the isolation of toxins from the blue-green alga, Microcystis aeruginosa forma aeruginosa, are described. A novel approach is reported, whereby contaminating impurities are succinylated, exploiting the absence of free amino groups in toxin variants. All toxin variants comprise a hydrocarbon blocking group, five amino acid residues detectable by conventional means, while methylamine is liberated upon acid hydrolysis. Possible structural features are discussed relating to the observed chemical and physical properties of the toxins.

Asunto(s)

RESUMEN

The venom of Atractaspis is unique in having a large percentage of both high and low molecular weight components. Its Sephadex G-50 S5 fraction appears to represent a new type of toxin that contains 17-18 Asx, 13-14 Cys and 10-11 Glx out of a total of 72-78 amino acids. The N-terminal of this toxin does not seem to resemble any of the known toxins. The overall lethal potency of the venom is very high; i.v. injections of 5 microgram of venom or 1 microgram of fractions S5 or S6 per mouse causes death within minutes. The results of the present study corroborate previous findings that suggested a separate grouping of the snakes genus Atractaspis at the subfamilial or familial level.

Asunto(s)

RESUMEN

The influence of pH and temperature on the kinetic properties of esterase E-II from Bitis gabonica venom has been studied. The pH profiles for the hydrolysis of N alpha-benzoyl-L-arginine ethyl ester showed that a group with pK approximately 7 must be ionized for activity. Measurement of N alpha-benzyl-L-arginine-p-nitroanilide hydrolysis reveals that the pK of the active site is significantly lowered (i.e. to approximately 6.3) in the enzyme-substrate complex. The temperature effect on the pK suggests the presence of a carboxylate in the active site of the enzyme. This suggestion is corroborated by the influence of organic solvent perturbations of the pK indicating a catalytic group of the neutral acid type. Values for the thermodynamic parameters associated with activation are reported. The transition state for ester degradation is at lower delta G and delta H levels than that for amide degradation. delta H and delta S for ester hydrolysis were noted to compensate each other with a compensation temperature Tc = 339K. Compensation can probably be related to weak bonding effects.

Asunto(s)

RESUMEN

The kinetics of arginine esterases E-I, E-II and E-III from the venom of Bitis gabonica were investigated. With N alpha-benzoyl-L-arginine ethyl ester as substrate linear competitive inhibition versus L-arginine was observed while ethanol gave rise to S-parabolic I-linear noncompetitive inhibition. Hydrolysis of N alpha-benzoyl-L-arginine-p-nitroanilide was noncompetitively inhibited by p-nitroaniline. Both slopes and intercepts of double reciprocal plots were a linear function of inhibitor concentration. Ethanol gave complex inhibition kinetics which could be interpreted in terms of mixed dead-end and alternate product inhibition (S-parabolic I-hyperbolic noncompetitive inhibition). These results imply an ordered uni-bi as the minimal kinetic mechanism wherein ethanol (or amine when amide is used as substrate) is released first from the enzyme surface, followed by the liberation of arginine. The enzymes are inactivated by phenylmethane sulfonyl fluoride which suggests the presence of an essential serine in the active sites of the enzymes. The enzymes may therefore be classified in the group of serine proteases.

Asunto(s)

RESUMEN

The Macrotyloma axillare plant, belonging to the Leguminosae family, is a perennial climbing or trailing herb 0.2--3.5 m long. The plant is indigenous to South Africa and it occurs in the warm dry northern parts of the Transvaal. It has been introduced into Australia, where the seed are used as animal food. Two protease inhibitors, DE-3 and DE-4, were purified from Macrotyloma axillare seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DEAE-cellulose. They each comprise 76 amino acid residues including 14 half-cystine residues. The complete primary structures of the two protease innibitors have been elucidated and their sequences are 67% identical. The inhibitor specificities, the sequences, the invariant amino acid residues and the reactive inhibitor sites of protease inhibitors DE-3 and DE-4 resemble the corresponding properties of the Bowman-Birk double-headed protease inhibitor group. The cysteine residues are in similar locations to those in protease inhibitors of known structure so they are presumed to link similarly.

Asunto(s)

Asunto(s)