Kinetic analysis of genipin degradation in aqueous solution.

Degradation of genipin (GP), a low toxicity natural protein crosslinking agent, in aqueous solution was monitored by HPLC at various pH levels. Degradation of GP was consistent with a mechanism consisting of a first order reaction with a reversible first step. Formation of the intermediate was slowest at more neutral pHs while formation of the irreversible product was correlated to increasing alkalinity.

Optimization of protein crosslinking formulations for the treatment of degenerative disc disease.

Study Design: Biochemical studies aimed at optimization of protein crosslinking formulations for the treatment of degenerative disc disease and subsequent biomechanical testing of tissues treated with these formulations.

Objective: To optimize protein crosslinking formulations for treatment of degenerating spinal discs.

Summary Of Background Data: Nonsurgical exogenous crosslinking therapy is a potential new, noninvasive technology for the treatment of degenerative disc disease.

Thermal analysis reveals differential effects of various crosslinkers on bovine annulus fibrosis.

Treatment of a pathological spinal disc in vivo by injection of protein crosslinking reagents to restore the disc's mechanical properties is a new approach to the treatment of degenerative disc disease. In this study, the thermal stability of the collagen in disc annulus was measured by differential scanning calorimetry following treatment with six different crosslinking agents. The crosslinkers used were; L-threose (LT), genipin (GP), methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), glutaraldehyde (GA), and proanthrocyanidin (PA).

Kinetic characterization and comparison of various protein crosslinking reagents for matrix modification.

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; L: -threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP >> LT.

Large disk intermediate precedes formation of apolipoprotein A-I-dimyristoylphosphatidylcholine small disks.

Small approximately 8.5 nm disks formed spontaneously when dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUVs) were incubated with apolipoprotein A-I (apoA-I) (100:1 molar ratio). However, in a time course study, the transient production of approximately 11 nm large disks was detected and isolated by gel filtration.

Fluorescence spectroscopic characterization of Humicola lanuginosa lipase dissolved in its substrate.

The conformational dynamics of Humicola lanuginosa lipases (HLL) and its three mutants were investigated by steady state and time-resolved fluorescence spectroscopy in two different media, aqueous buffer and the substrate triacetin. The fluorescence of the four Trps of the wild-type HLL (wt) reports on the global changes of the whole lipase molecule. In order to monitor conformational changes specifically in the alpha-helical surface loop, the so-called 'lid' of HLL comprised of residues 86-93, the single Trp mutant W89m (W117F, W221H, W260H) was employed.

The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain.

Phe(1395) stacks parallel to the FAD isoalloxazine ring in neuronal nitric-oxide synthase (nNOS) and is representative of conserved aromatic amino acids found in structurally related flavoproteins. This laboratory previously showed that Phe(1395) was required to obtain the electron transfer properties and calmodulin (CaM) response normally observed in wild-type nNOS. Here we characterized the F1395S mutant of the nNOS flavoprotein domain (nNOSr) regarding its physical properties, NADP(+) binding characteristics, flavin reduction kinetics, steady-state and pre-steady-state cytochrome c reduction kinetics, and ability to shield its FMN cofactor in response to CaM or NADP(H) binding.

Interactions among membrane and soluble components of the flagellar export apparatus of Salmonella.

Interactions among several components of the flagellar export apparatus of Salmonella were studied using affinity chromatography, affinity blotting, and fluorescence resonance energy transfer (FRET). The components examined were two integral membrane proteins, FlhA and FlhB, and two soluble components, FliH and the ATPase FliI. Affinity chromatography and affinity blotting demonstrated a heterologous interaction between FlhA and FlhB but not homologous FlhA-FlhA or FlhB-FlhB interactions.