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We have previously described the development of cloning vectors for the production of OprI-based outer membrane fusion proteins in E. coli (Cornelis et al., 1996) and now describe the construction of a new vector, containing a lacI(q) gene, resulting in tight repression of the promotor and allowing its use in other Gram (-) bacteria. The new pVUB3 expression vector encodes a truncated but active LacI(q)(341) repressor which binds to the single operator in the vector. A high repression of the trc promotor was observed, resulting in a very low basal leakage of expression and very high production levels of OprI or derivatives after IPTG induction in E. coli. Bacterial viability was not affected under uninduced conditions, but the number of viable cell counts decreased after production of large amounts of the outer membrane-bound OprI lipoprotein and its derivatives, both in E. coli and Salmonella typhimurium. This highly repressible system allows us to extend the use of OprI vectors in other Gram (-) bacteria, resulting in the production of outer membrane-bound lipid-modified molecules, opening the possibility for its application in the design of potential live Salmonella-based subunit vaccines.

Several pituitary hormones, including corticotropin (ACTH), growth hormone (GH), prolactin, and beta-endorphin (but not thyrotropin, follicle-stimulating hormone, or luteinizing hormone), are released in response to hypoglycemia in normal subjects. In patients with insulin-dependent diabetes mellitus (IDDM), the degree of glycemic control is known to alter ACTH and GH responses to hypoglycemia. The current study was performed to examine the effect of glycemic control on prolactin and beta-endorphin responses to hypoglycemia in subjects with IDDM. We performed 3-hour stopped hypoglycemic-hyperinsulinemic clamp studies (12 pmol/kg/min) during which plasma glucose was decreased from 5.0 mmol/L to 2.2 mmol/L in steps of 0.6 mmol/L every 30 minutes in 20 subjects with uncomplicated IDDM (12 males and eight females; age, 26 +/- 2 years; IDDM duration, 10 +/- 1 years; body mass index, 23.6 +/- 0.6 kg/m2) and 10 healthy subjects (five males and five females aged 30 +/- 1 years). The 10 diabetic subjects in good glycemic control (mean hemoglobin A1 [HbA1], 7.5% +/- 0.3%; normal range, 5.4% to 7.4%) were compared with the 10 poorly controlled patients (mean HbA1, 12.6% +/- 0.5%; P.001 v well-controlled diabetic group). During hypoglycemia, prolactin levels in the well-controlled diabetic group did not change (7 +/- 1 microgram/L at plasma glucose 5.0 mmol/L to 9 +/- 2 micrograms/L at plasma glucose 2.2 mmol/L), whereas prolactin levels increased markedly in the poorly controlled diabetic group (7 +/- 2 micrograms/L to 44 +/- 17 micrograms/L) and healthy volunteers (12 +/- 2 micrograms/L to 60 +/- 19 micrograms/L, P.05 between IDDM groups). The plasma glucose threshold required for stimulation of prolactin secretion was 2.2 +/- 0.1 mmol/L in well-controlled IDDM, 3.0 +/- 0.4 mmol/L in poorly controlled IDDM, and 2.4 +/- 0.1 mmol/L in healthy subjects (P.05 between IDDM groups). Responses in males and females were similar. The increase in beta-endorphin levels was also attenuated in well-controlled IDDM patients (4 +/- 1 pmol/L at plasma glucose 5.0 mmol/L to 11 +/- 4 pmol/L at plasma glucose 2.2 mmol/L) versus poorly controlled IDDM patients (5 +/- 1 pmol/L to 26 +/- 7 pmol/L) and healthy subjects (8 +/- 1 pmol/L to 56 +/- 13 pmol/L). The plasma glucose threshold required for stimulation of beta-endorphin release was again lower in well-controlled IDDM versus poorly controlled IDDM patients (2.2 +/- 0.1 v 3.0 +/- 0.3 mmol/L) and healthy subjects (2.5 +/- 0.4 mmol/L, P.05 between IDDM groups). In conclusion, prolactin and beta-endorphin responses to a standardized hypoglycemic stimulus (plasma glucose, 2.2 mmol/L) are reduced and plasma glucose levels required to stimulate release of prolactin and beta-endorphin are lower in well-controlled IDDM compared with poorly controlled IDDM and healthy subjects. Thus, stress hormones not previously considered to have a primary role in plasma glucose recovery from hypoglycemia are affected by glycemic control, suggesting a more generalized alteration of hypothalamic-pituitary responses to hypoglycemia in IDDM patients with strict glycemic control.