Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine.

We have increased the methionine content of the seed proteins of a commercial winter variety of canola by expressing a chimeric gene encoding a methionine-rich seed protein from Brazil nut in the seeds of transgenic plants. Transgenic canola seeds accumulate the heterologous methionine-rich protein at levels which range from 1.7% to 4.0% of the total seed protein and contain up to 33% more methionine. The precursor of the methionine-rich protein is processed correctly in the seeds, resulting in the appearance of the mature protein in the 2S protein fraction. The 2S methionine-rich protein accumulates in the transgenic seeds at the same time in development as the canola 11S seed proteins and disappears rapidly upon germination of the seed. The increase in methionine in the canola seed proteins should increase the value of canola meal which is used in animal feed formulations.

Informed consent: can we educate patients?

Previous reports have shown consent to be 'short' of informed in many cases. One hundred patients, including a control group, were interviewed between 2 and 7 days after their operations. Those given an information sheet before the operation had a better recall of their operations than the controls, the overall awareness being 97 and 69% respectively. This may have medico-legal implications but further studies are required.

Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants.

We have constructed a chimeric gene encoding a Brazil nut methionine-rich seed protein which contains 18% methionine. This gene has been transferred to tobacco and expressed in the developing seeds. Tobacco seeds are able to process the methionine-rich protein efficiently from a larger precursor polypeptide of 17 kDa to the 9 kDa and 3 kDa subunits of the mature protein, a procedure which involves three proteolytic cleavage steps in the Brazil nut seed. The accumulation of the methionine-rich protein in the seeds of tobacco results in a significant increase (30%) in the levels of the methionine in the seed proteins of the transgenic plants. Our data indicate that the introduction of a chimeric gene encoding a methionine-rich seed protein into crop plants, particularly legumes whose seeds are deficient in the essential sulfur-containing amino acids, represents a feasible method for improving the nutritional quality of seed proteins.

Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine.

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.

Automated enzyme assays by use of a centrifugal analyzer with fluorescence detection.

The first commercially available centrifugal analyzer having fluorescence detection capability was used to develop kinetic fluorometric assays for several proteinases. The substrates were all synthetic oligopeptides incorporating 7-amino-4-trifluoromethylcoumarin, a molecule that can be detected either spectrophotometrically or fluorometrically. We thus compared the centrifugal analyzer spectrophotometric and fluorometric detection systems, finding fluorescence detection to be 50-fold more sensitive. We also compared the sensitivity of the fluorescence detector to that of a conventional spectrofluorometer by determining the minimum detection limit for each enzyme on both instruments; we found them to be similar in sensitivity. As an illustrative application, we measured the cathepsin B-like activity in serum samples from 55 women. The median enzyme activity of women taking oral contraceptives and pregnant women was increased two- and threefold, respectively, over the control group (about 5% CV within run, and 10% CV between runs).

Revagotomy for recurrent peptic ulceration.

A review has been made of 59 patients with recurrent peptic ulceration after incomplete vagotomy. Eighteen transthoracic procedures were performed, the remainder having abdominal revagotomy. An antrectomy was also carried out in 10 of these patients. An intact posterior nerve trunk was the most common operative finding and when either an anterior trunk or nerve strands only were present there was less likely to be an early positive insulin response (P = 0.033). Following incomplete vagotomy a longer period of symptomatic relief is obtained when gastro-enterostomy rather than pyloroplasty is used as the drainage (P less than 0.01). Completing the vagotomy by the abdominal route gave superior results to transthoracic revagotomy (P = 0.0015), the former procedure without antrectomy having no associated mortality in this series. Although the results of revagotomy and antrectomy are as good as those of transabdominal revagotomy alone, we recommend the latter more conservative treatment for recurrent ulceration after incomplete vagotomy.

Effects of partial surgical pancreatectomy in rats. I. Pancreatic regeneration.

The capacity for pancreatic regeneration in male Wistar rats of average weight 176 g was determined after surgical resection of 50, 70, or 90% of the pancreas. In control animals of this weight pancreatic growth was still taking place but ceased after 2 months. Growth of the residual pancreas in resected animals over this period was greater than in controls and the final weight of the residual pancreas was 21, 32, and 78% greater than that of the corresponding segments in sham-operated animals after 50, 70, and 90% resection respectively. Proliferation of acinar cells was assayed by autoradiographic detection of tritiated thymidine incorporation in controls and after 90% resection. Proliferation of acinar cells in control animals during the first 2 weeks of the experiment was significantly higher than at later times when it fell to very low levels. Proliferation in 90% resected animals was markedly higher than in controls from 2 to 5 days after resection and, together with the limited hypertrophy occurring at this time, adequately accounted for the increase in weight of the residual pancreas during the first 2 weeks. We were unable to explain the increase in pancreatic weight in control or resected animals between 2 weeks and 2 months either in terms of hypertrophy or hyperplasia of acinar cells.