NMR spectroscopy in beta cell engineering and islet transplantation.

Islet transplantation is a promising method for restoring normoglycemia and alleviating the long term complications of diabetes. Widespread application of islet transplantation is hindered by the limited supply of human islets and requires a large increase in the availability of suitable insulin secreting tissue as well as robust quality assessment methodologies that can ensure safety and in vivo efficacy. We explore the application of nuclear magnetic resonance (NMR) spectroscopy in two areas relevant to beta cell engineering and islet transplantation: (1) the effect of genetic alterations on glucose metabolism, and (2) quality assessment of islet preparations prior to transplantation. Results obtained utilizing a variety of NMR techniques demonstrate the following: (1) Transfection of Rat1 cells with the c-myc oncogene (which may be involved in cell proliferation and cell cycle regulation) and overexpression of Bcl-2 (which may protect cells from stresses such as hypoxia and exposure to cytokines) introduce a wide array of alterations in cellular biochemistry, including changes in anaerobic and oxidative glucose metabolism, as assessed by 13C and 31P NMR spectroscopy. (2) Overnight incubation of islets and beta cells in the bottom of centrifuge tubes filled with medium at room temperature, as is sometimes done in islet transportation, exposes them to severe oxygen limitations that may cause cell damage. Such exposure, leading to reversible or irreversible damage, can be observed with NMR-detectable markers using conventional 13C and 31P NMR spectroscopy of extracts. In addition, markers of irreversible damage (as well as markers of hypoxia) can be detected and quantified without cell extraction using high-resolution magic angle spinning 1H NMR spectroscopy. Finally, acute ischemia in a bed of perfused beta cells leads to completely reversible changes that can be followed in real time with 31P NMR spectroscopy.

Glucose-stimulated insulin secretion is not obligatorily linked to an increase in O2 consumption in betaHC9 cells.

We investigated the effects of glucose on the rates of oxygen consumption (OCR) and insulin secretion (ISR) by betaHC9 cells derived from mouse pancreatic islets with beta-cell hyperplasia. Our results demonstrate that the OCR by betaHC9 cells incubated in nutrient-rich DMEM is unaffected by glucose (0-25 mM), is dissociated from the ISR (which increases with the addition of glucose), and is always higher than that measured in PBS. Glucose (25 mM) increases both the OCR and ISR when added to nutrient-free PBS. On the basis of results presented here, we suggest that, contrary to the current consensus, the observed increases in the OCR by beta-cells upon addition of glucose to nutrient-free buffers may be unrelated to the process of glucose-stimulated insulin secretion (GSIS) and, instead, related to nutrient starvation. We believe that a reevaluation of the implication of changes in OCR upon glucose stimulation in the process of GSIS is warranted and that OCR and ISR measurements should be performed in more physiological media to avoid nutrient starvation artifacts.

Study of coumarin metabolism by Chinese hamster lung fibroblasts expressing a human cytochrome P450 using H-nmr.

1. Human cytochrome P450 2A6 is expressed in Chinese hamster lung fibroblasts. The isozyme hydroxylates coumarin at the 7-position. 2. Metabolism of coumarin in these lung fibroblasts was monitored using 1H-nmr. Media samples were taken from cells grown in flasks and also in a fluidized bed bioreactor. In each case 7-hydroxycoumarin was readily observable by nmr in crude extracts of the medium. 3. The rate of formation of 7-hydroxycoumarin observed in the acute bioreactor experiments on a per cell basis was found to be much higher than that obtained under chronic monolayer conditions, and correlated with glucose consumption rates.

Structures of paralytic acylpolyamines from the spider Agelenopsis aperta.

The structures are given for five paralytic acylpolyamines from the venom of the funnel web spider, Agelenopsis aperta. The acyl moieties are derived from (3-indolyl)acetic acid, (4-hydroxy-3-indolyl)acetic acid, and 4-hydroxybenzoic acid. The polyamine portions of the toxins are novel. Three toxins (AG489, AG505, and AG452) contain 1, 5, 9, 13, 18, 22-hexaazadocosane which is unique as a natural polyamine because of its length and hydroxylation at the 5-aza position. The polyamine portions of two other alpha-agatoxins (AG488 and AG504) are unusual also, containing guanidinooxy moieties.

Structures and biological activities of three synaptic antagonists from orb weaver spider venom.

The venom of Argiope aurantia, an orb weaver spider, contains a mixture of low molecular weight "argiotoxins", which block neuromuscular transmission in insects. Complete structure elucidation of three argiotoxins reveals common features; a hydrophilic, basic domain of arginine, a polyamine and asparagine is connected to an aromatic moiety contributed either by 4-hydroxyindole-3-acetic acid or 2,4-dihydroxyphenylacetic acid. Structural assignments of two argiotoxins are verified by chemical synthesis. The argiotoxins cause reversible paralysis when injected into insects and this is correlated with a stimulus-dependent inhibition of skeletal neuromuscular transmission at submicromolar concentrations.

lac repressor-lac operator interaction: NMR observations.

We show here the changes in the NMR spectra of the Escherichia coli lac repressor when bound to isolated lac operator DNA. The observations focus on the aromatic residues--four tyrosines and a single histidine--in the amino-terminal DNA binding domain of the lac repressor. There is a good correlation between chemical shift changes seen by 19F NMR when compared with 1 H NMR of otherwise identical repressor--DNA complexes. The results suggest that the tyrosines do not intercalate in the DNA. The NMR spectral changes with similarly sized DNA fragments, not containing the lac operator DNA sequence, are different. Thus, the amino-terminal domain of the lac repressor is independently capable of discriminating between lac operator and nonspecific DNA sequences. There can be two amino-terminal fragments per operator in the specific complex.

Inducer and anti-inducer interactions with the lac repressor seen by nuclear magnetic resonance changes at tyrosines and tryptophans.

The effects of binding inducer and anti-inducer of the Escherichia coli lac operon to the lac repressor were examined by taking advantage of fluorine-19 NMR. The fluorine nucleus was biosynthetically incorporated into the lac repressor with either 5-fluorotryptophan or 3-fluorotyrosine. It is suggested that these small effector molecules influence the operator-binding properties of the tetrameric lac repressor by altering the intersubunit relationships in the protein.

Genetic insertion of nuclear spin labels in the lac repressor.

A general strategy for the insertion of nuclear spin labels throughout the sequence of a protein is illustrated with the Escherichia coli lac repressor. Examples are shown where the 19F nucleus is incorporated using 3-fluorotyrosine, as well as the selective insertion of additional protons. These selectively inserted nuclei give additional resonances in the respective NMR spectra that can be used to probe the structure and function of the proteins.

Genetic assignment of resonances in the NMR spectrum of a protein: lac repressor.

By using a systematic genetic approach, the resonances in the 19F NMR spectrum of 3-fluorotyrosine-substituted lac repressor protein have been assigned. The NMR data indicate that each monomer of the repressor consists of two distinct and independent domains. One domain, the NH2-terminal sixth of the primary sequence, which has been shown to be very important for DNA binding, is very mobile. The remaining COOH-terminal sequence is more rigid. Ligands of the repressor, which affect its DNA binding capability, lead to conformational changes in the COOH-terminal domain. The approach to the assignment of spectral features taken here can be extended to other systems.