Double-shot magnetic resonance imaging of cerebral lesions: fast spin-echo versus echo planar sequences.

The authors compared two new rapid MRI techniques: double-shot echo-planar imaging (DS-EPI) versus double-shot fast spin-echo (DS-FSE) in the evaluation of cerebral lesions. The authors examined 35 patients with 37 lesions, which were hyperintense on long TR images. Patients were scanned with both DS-EPI and DS-FSE with a time of repetition (TR) of 10,000 milliseconds and an echo time (TE) of 80 milliseconds. Conspicuity was determined from region of interest measurements to calculate contrast to noise ratio (C/N). Visual comparisons between DS-EPI and DS-FSE, and between DS-EPI and T2-weighted conventional spin-echo (CSE) were also performed to evaluate the sequences' ability to depict hemorrhage. The mean C/N for both sequences was comparable: 36.7 for DS-FSE and 35.6 for DS-EPI, with no statistically significant difference (p = 0.77). With regards to depicting blood products, DS-EPI proved far more effective than DS-FSE and comparable to CSE. Also, DS-EPI proved to be more time-efficient, requiring 1.67 seconds per section, while DS-FSE required 3.33 seconds per section. Whereas DS-FSE and DS-EPI are comparable in their ability to depict hyperintense cerebral pathology, DS-EPI is more time-efficient, and therefore appears preferable. Because of the high magnetic susceptibility of DS-EPI, geometric distortion degrades visualization of lesions in the posterior fossa or near the sinuses. On the other hand, the high magnetic susceptibility results in high conspicuity of blood products.

Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts with aliphatic thiols. A new thiolation procedure for proteins is based on this reagent. The procedure involves two steps. First, 2-pyridyl disulphide structures are introduced into the protein by the reaction of some of its amino groups with the N-hydroxysuccinimide ester sie of the reagent. The protein-bound 2-pyridyl disulphide structures are then reduced with dithiothreitol. This reaction can be carried out without concomitant reduction of native disulphide bonds. The technique has been used for the introduction of thiol groups de novo into ribonuclease, gamma-globulin, alpha-amylase and horseradish peroxidase. N-Succinimidyl 3-(2-pyridyldithio)propionate can also be used for the preparation of protein-protein conjugates. This application is based on the fact that protein-2-pyridyl disulphide derivatives (formed from the reaction of non-thiol proteins with the reagent) react with thiol-containing proteins (with native thiols or thiolated by, for example, the method described above) via thiol-disulphide exchange to form disulphide-linked protein-protein conjugates. This conjugation technique has been used for the preparation of an alpha-amylase-urease, a ribonuclease-albumin and a peroxidase-rabbit anti-(human transferrin) antibody conjugate. The disulphide bridges between the protein molecules can easily be split by reduction or by thiol-disulphide exchange. Thus conjugation is reversible. This has been demonstrated by scission of the ribonuclease-albumin and the alpha-amylase-urease conjugate into their components with dithiothreitol. N-Succinimidyl 3-(2-pyridyldithio)propionate has been prepared in crystalline form, in which state (if protected against humidity) it is stable on storage at room temperature (23 degrees C).

Purification of uricase by biospecific adsorption-desorption.

A simple procedure for the purification of uricase from bovine kidney is described. The procedure involves the following steps: 1) processing of kidney mince by borate/butanol, 2) ammonium sulphate precipitation, and 3) biospecific adsorption-desorption. The adsorbents were prepared by chemical attachment of urate or xanthine to agarose gel beads. The desorption was performed by a xanthine solution. The adsorption-desorption procedure resulted in an 11 000-12 000-fold purification. The specific activity of the purified uricase was 19.8 U/mg using either "urate" adsorbent. The recovery was about 70%. The adsorbents were also used for the purification of commercial uricase preparations from hog liver. In this case the purified uricase also possessed a specific activity of 19.8 U/mg. The products were homogenous as judged by gradipore electrophoresis and gel filtration.

Immobilization of enzymes based on hydrophobic interaction. I. Preparation and properties of a beta-amylase adsorbate.

Sweet potato beta-amylase (alpha-1,4 glucan maltohydrolase, EC 3.2.1.2) was immobilized through adsorption onto an agrose gel to which nonpolar side chains had been introduced via ether bridges. The adsorbent showed evidence of saturation at an enzyme content of 35 mg per milliliter of packed gel. The adsorption was rapid and yielded a product whose operational stability depended on the initial content of beta-amylase. Activity leakage was low. The relative activity of immobilized enzyme was inversely related to the amount of enzyme adsorbed to a given gel volume, having a maximal value of around 50% at low enzyme contents.

Immobilization of enzymes based on hydrophobic interaction. III. Adsorbent substituent density and its impact on the immobilization of beta-amylase.

Hexyl-groups have been introduced into crosslinked Sepharose 6B, yielding gels with degrees of substitution which range from 0.02 to 0.70 mol hexyl-side chain per mole galactose residue. The gels were exposed to beta-amylase in solution, and the resulting adsorbates indicated a monotonic increase in adsorption capacity with an increasing hexyl-content. Adsorbate activity, by contrast, displayed a maximum for a carrier gel with a hexyl-galactose ratio of 0.51. Adsorbates based on gels with different hexyl-content were used in column reactors for continuous maltose production from a soluble starch substrate.

Immobilization of enzymes based on hydrophobic interaction. II. Preparation and properties of an amyloglucosidase adsorbate.

Amyloglucosidase from Aspergillus niger (alpha-1,4 and 1,6 glucan glucohydrolase, EC 3.2.1.3) was immobilized through adsorption onto a hexyl-Sepharose, containing 0.51 mol hexyl-group per mole of galactose. The adsporption limit of the carrier with respect to this enzyme was about 17 mg per gram wet conjugate. The retention of activity upon immobilization was high, varying from essentially full activity at low enzyme content down to 68% at the adsorption limit. The immobilized preparation, as well as the soluble enzyme, showed apparent zero order kinetics within 60% of the substrate's conversion limit. Product inhibition of the soluble enzyme showed a KI of 5-10(-2)M. In the presence of 3M NaCl, adsorbates were formed more rapidly and with a higher yield of immobilized protein, but with lower specific activity. Conjugates resulting from adsorption of amyloglucosidase in identical concentrations, but at different salt contents, showed comparable activities and operational stabilities. Continuous operation from three months reduced conjugate activity to 40%. The thermal stability of the adsorbate was inferior to that of the soluble enzyme, but was noticeably enhanced in the presence of substrate.

Reversible, covalent immobilization of enzymes by thiol-disulphide interchange.

1. alpha-Amylase and alpha-chymotrypsin have been immobilized by covalent attachment to mercaptohydroxypropyl ether agarose gel. The technique involves two steps: (a) thiolation of the enzymes by methyl 3-mercaptopropioimidate, (b) coupling of the thiolated enzymes to a mixed disulphide derivative of agarose obtained by reacting mercaptohydroxypropyl ether agarose with 2,2'-dipyridyl disulphide. 2. The immobilization technique can be performed so that most of the inherent activity of the enzymes is conserved. However, diffusion limitations and steric factors prevent full manifestation of the immobilized activities. 3. Immobilized alpha-amylase was used in a packed-bed reactor for the continuous hydrolysis of starch. When the enzymically active gel had lost its activity it could be regenerated in situ by reductive uncoupling of the inactive protein and attachment of a new portion of thiolated alpha-amylase.