Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000

Oral delivery of insulin to diabetic patients is highly desirable because it would be non-invasive and more closely mimic normal physiology, but this route of administration typically results in low bioavailability due to low pH and enzymatic degradation along the gastrointestinal tract. To explore an alternative approach that may mitigate these obstacles and also facilitate local synthesis of new therapeutic protein molecules in the small intestine, we engineered the food-grade bacterium Lactococcus lactis (NZ9000) for nisin-inducible expression and secretion of a bioactive single-chain insulin (SCI) analog, SCI-57. We show that the addition of nisin during early-log phase has a modest inhibitory effect on cell growth but induction during mid-log phase has a negligible impact on proliferation, suggesting a tradeoff between cell growth rate and duration of induction. We find that a signal peptide such as usp45 is necessary for secretion of SCI-57 into the medium; furthermore, we demonstrate that this secreted SCI-57 is biologically active, as assessed by the ability of conditioned L. lactis medium to stimulate Akt signaling in differentiated 3T3-L1 adipocytes. Finally, we show that the biological activity of SCI-57 was enhanced by near-neutral or slightly alkaline pH during induction, which is comparable to the pH in the small intestine, and by removal of a C-terminal purification tag. This study demonstrates that food-grade bacteria can be engineered to secrete bioactive insulin analogs and opens up the possibility of oral insulin delivery using live microorganisms.     

Reductive methylation of insulin. Production of a biologically active tritiated insulin

Reductive methylation of the three amino groups of porcine insulin was accomplished by incubation with formaldehyde and sodium cyanoborohydride. The two amino termini and the epsilon amino group of B29 lysine were each dimethylated within 1 h of incubation. The fully methylated insulin bound more tightly to a reverse phase column than did native insulin, had a slightly more acid isoelectric point, and maintained approximately 50% biological activity when examined with an insulin sensitive cultured cell line. Reductive methylation with sodium cyanoboro [3H] hydride resulted in a [3H] methylated insulin with a specific activity of 6 Ci/mmol.     

Neurotensin, a biologically active peptide.

Neurotensin, a tridecapeptide appears to be localized in various parts of the brain and gut. A high affinity binding component of neurotensin to brain membranes, synaptosomes and mast cells has been reported. After peripheral administration the peptide exerts a medley of effects which appear directed mainly to glucose metabolism. In addition, complex vascular effects have also been noted including hypotensin, cyanosis, vasodilation and increased permeability. The peptide may also be associated with inflammatory events. Complex effects upon the secretion of anterior pituitary tropic hormones have been observed.After central administration neurotensin exerts several effects all of which appear to be sufficiently explained by the potent hypothermic action. The resolution of the question of which, if any, of the actions of neurotensin are involved in physiological regulation has not been achieved.     

Secretory expression and surface display of a new and biologically active single-chain insulin (SCI-59) analog by lactic acid bacteria

Applied Microbiology and Biotechnology - Insulin plays an important role in drug therapies for diabetes mellitus and as the main route of insulin delivery, subcutaneous injection may cause local...     

Dynamics of biologically active enterotoxin release by E. coli]

Accumulation of E. coli enterotoxin in the Finkelshtein's culture medium in growing the cells in a 30-litre reactor was studied. Accumulation of active highly molecular enterotoxin occurred in the course of a 6-hour cultivation of E. coli, strain P-99 (O141: K85ab; K88ab: H4) in the fluid medium under aeration. Oxygen utilization, synthesis and release into the nutrient medium of pyruvic acid, and protein accumulation were observed. The preparation obtained was stable to the lyophylic drying, contained thermolabile and thermostable toxins and marked edema of mouse limbs. The data obtained were of significance for the industrial production of active enterotoxin preparations.     

LysB3, GluB29] insulin: a novel insulin analog with enhanced beta-cell protective action

Insulin receptor substrate (IRS)-2 has been implicated in the promotion of beta-cell survival. Here we tested the hypothesis that the novel analog [LysB3, GluB29] insulin (insulin glulisine, IG) might mediate an enhanced beta-cell protective effect due to its unique property of preferential IRS-2 phosphorylation. We assessed IRS activation by IG and its anti-apoptotic activity against cytokines or palmitic acid in comparison to insulin, insulin analogs, and insulin-like growth factor (IGF)-I using INS-1 cells. IG induced a prominent IRS-2 activation without significant IRS-1 stimulation. The marked cytokine- and fatty acid-induced apoptosis was strongly (55-60%) inhibited by IG both at the level of caspase 3 activation and nucleosomal release, with only 15% inhibition of apoptosis by regular insulin. At 1nM, insulin, insulin aspart, and insulin lispro were much less effective compared to IG. In conclusion, the prominent anti-apoptotic activity of insulin glulisine might serve to counteract autoimmune- and lipotoxicity-induced beta-cell destruction.     

Lipotropin: precursor to two biologically active peptides.

Lipotropin appears to be the common precursor to β-MSH, a peptide with lipolytic activity, and C-Fragment, a peptide with potent opiate activity. The product formed is determined by the specificity of the activating enzymes.The amino acid sequence of β-MSH, the 18 residue melanocyte stimulating hormone, is contained within the central region of lipotropin (LPH), a 91 residue polypeptide. On this basis Li and his colleagues¹ suggested that LPH might be the prohormone of β-MSH. Bertagna, Lis and Gilardeau², on the other hand, were unable to demonstrate conversion of LPH to β-MSH $\text{in vitro}$ using pulse labelling techniques. If LPH is the precursor of β-MSH, formation of the hormone should be accompanied by release of the contiguous fragments of the prohormone and the fragments remain in the secretory particle of the gland. To obtain evidence on the biosynthetic origin of β-MSH, we have isolated peptides from pituitary in a search for the N- and C-fragments of the prohormone.     

Conformational analysis of the biologically active cyclic analog of beta-casomorphin H-Tyr-cyclo[D-OrnPheProGly].

A biologically active analog of beta-casomorphin, H-Tyr-cyclo[D-OrnPheProGly], was studied by theoretical conformational analysis. Random sampling was used to search the conformational space of allowed dihedral angles. Among 53 low-energy conformers with different folding of the peptide cyclic moiety, only those were selected which correspond to the low-energy area of the model linear tripeptide Ac-D-AlaAlaPro-NHMe. This peptide was used as the main chain "template" for the D-OrnPheProGly fragment of the studied cyclopeptide molecule. Only 15 conformers were chosen as potentially biologically active structures. The conformational possibilities of the Phe residue were constrained to the combined (A + G) region of the Ala residue phi,psi-map for linear peptides.     

Transfer factor: isolation of a biologically active component

The chromatographie separation of transfer factor from a tuberculin-sensitive donor resulted in isolation of a fraction which was proved to be active by both local and systemic transfer of the specific immunity present in the donor. This isolation technique may lead to the ultimate characterization of transfer factor.     

Synthesis of biologically active porcine secretin and [ITyr10] porcine secretin.

Porcine secretin, [Tyr10] secretin, and [Tyr13] secretin were synthesized by solid phase methodology and purified by stepwise gradient elution from a short reversed-phase column with ethanol and acetic acid as organic modifiers. [Tyr10] secretin and [Tyr13] secretin were iodinated by the chloramine-T method and nonmono-, and di-iodinated products separated and isolated by reversed-phase HPLC. Batch incubation analysis is isolated mouse pancreatic islets revealed that secretin and the [Tyr10] analogue were indistinguishable in their effect on the glucose-induced insulin release and cAMP accumulation. [Tyr13] secretin in contrast was significantly less potent in its effect on the glucose-induced insulin release.     

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-"glyburide", has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values of 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).     

The amylin superfamily: A novel grouping of biologically active polypeptides related to the insulin A-chain

The peptide amylin (previously termed Diabetes Associated Peptide) has recently been isolated and characterised from the amyloid of the pancreatic islets of Langerhans from human type 2 diabetics [1]. Amylin shows about 46% identity in amino acid sequence on comparison with the calcitonin gene-related peptides (CGRPs) and also shows some similarity to insulin [1]. Recent studies have also shown that both amylin and CGRP are potent inhibitors of insulin-stimulated glycogen synthesis in skeletal muscle in vitro [2,3]. Hormones may be arranged into families, therefore a degree of order exists even though hormone-mediated effects are complex [4]. The polypeptides insulin, insulin-like growth factors (IGFs) and relaxins have been grouped into such a family with similarities both at the protein-structural and genetic levels [4,5]. We now demonstrate that this insulin-related family, along with amylin and the CGRPs, are members of a peptide superfamily defined by structural similarity in the region corresponding to the A-chain of insulin. In order to distinguish this grouping of small biologically active peptides from the previous one, we have designated it the amylin superfamily. All the members of the previously defined insulin family have a region homologous to the insulin B-chain. Insulin, the IGFs, the relaxins, the CGRPs and amylin are all involved in carbohydrate metabolism and therefore these peptides are functionally as well as structurally related. This grouping of peptides may have important implications for the study of human metabolic disease.