Determination of reference values for optical properties of liquid phantoms based on Intralipid and India ink.

A multi-center study has been set up to accurately characterize the optical properties of diffusive liquid phantoms based on Intralipid and India ink at near-infrared (NIR) wavelengths. Nine research laboratories from six countries adopting different measurement techniques, instrumental set-ups, and data analysis methods determined at their best the optical properties and relative uncertainties of diffusive dilutions prepared with common samples of the two compounds. By exploiting a suitable statistical model, comprehensive reference values at three NIR wavelengths for the intrinsic absorption coefficient of India ink and the intrinsic reduced scattering coefficient of Intralipid-20% were determined with an uncertainty of about 2% or better, depending on the wavelength considered, and 1%, respectively. Even if in this study we focused on particular batches of India ink and Intralipid, the reference values determined here represent a solid and useful starting point for preparing diffusive liquid phantoms with accurately defined optical properties. Furthermore, due to the ready availability, low cost, long-term stability and batch-to-batch reproducibility of these compounds, they provide a unique fundamental tool for the calibration and performance assessment of diffuse optical spectroscopy instrumentation intended to be used in laboratory or clinical environment. Finally, the collaborative work presented here demonstrates that the accuracy level attained in this work for optical properties of diffusive phantoms is reliable.

Special considerations concerning regulatory requirements and drug development for peptides and biotech products in the EU.

The marketing authorization for a new medicinal product is based on the scientific assessment of its quality, safety and efficacy. The marketing authorization application (MAA) which covers all the relevant documentation can be filed in the EU via different application procedures. For peptides and biological products special issues have to be taken into consideration during drug development. Due to special production procedures and the complexity of the active substance itself, peptides and biotech products are subject to specific regulatory requirements. This leads to the necessity to discuss the development program of a new peptide or biotech product with the health authorities on a case by case basis. This article will focus on the special regulatory requirements for peptides and biotech products including the registration procedures as well as technical, preclinical and clinical issues.

Molecular analysis of an anion pump: purification of the ArsC protein.

The ars operon of resistance plasmid R773 encodes an anion-translocating ATPase which catalyzes extrusion of the oxyanions arsenite, antimonite, and arsenate, thus providing resistance to the toxic compounds. Although both arsenite and arsenate contain arsenic, they have different chemical properties. In the absence of the arsC gene the pump transports arsenite and antimonite, oxyanions with the +III oxidation state of arsenic or antimony. The complex neither transports nor provides resistance to arsenate, the oxyanion of the +V oxidation state of arsenic. The arsC gene encodes a 16-kDa polypeptide, the ArsC protein, which alters the substrate specificity of the pump to allow for recognition and transport of the alternate substrate arsenate. The arsC gene was cloned behind a strong promoter and expressed at high levels. The ArsC protein was purified and crystallized.

Mutant proteins of human interleukin 2. Renaturation yield, proliferative activity and receptor binding.

Muteins, i.e. proteins altered by mutation of their genes, of interleukin 2 (Il2) were generated by oligonucleotide-directed mutagenesis in vitro. All acidic and basic residues conserved between man and mouse were exchanged as well as four lipophilic residues contained within four hydrophobic segments of the protein. The muteins were produced in Escherichia coli and submitted to a renaturation and purification protocol, before bioactivity and receptor binding of each of them was determined. All muteins besides two (K44/T125 and Q110/T125) could be renatured and purified. One mutein (K94/T125) exhibited a more than tenfold-improved renaturation yield. One amino exchange (Asp-20 to Asn) resulted in an about 20-fold reduction in proliferative activity and high-affinity receptor binding. The binding to the low-affinity Il2-binding protein (Tac antigen) was unimpaired. A second exchange (Arg-38 to Gln) had no effect on proliferative activity. The binding to both the high- and the low-affinity receptor, however, was reduced about 20-fold. Preliminary trials on the stability of these muteins by guanidinium hydrochloride denaturation studies detected no differences between wild-type interleukin 2 and muteins. It is suggested that Asp-20 forms part of the binding site for the large receptor subunit whereas Arg-38 is involved in the contact site to the small subunit.

Molecular characterization of an anion pump. The arsA gene product is an arsenite(antimonate)-stimulated ATPase.

The products of the arsenical resistance operon of resistance plasmid R733 form an efflux system for arsenicals. Detoxification results from active efflux of the oxyanions, preventing their concentration from reaching toxic levels. The largest polypeptide encoded by the ars operon was purified. From N-terminal sequencing the purified protein, termed the ArsA protein, was shown to correspond to the product of the arsA gene. The purified protein was demonstrated to bind ATP by two methods. First, a photoadduct of the protein with [alpha-32P]ATP was formed by irradiation at 254 nm. Second, the purified protein bound a fluorescent ATP analogue, 2',3'-o-(2,4,6)trinitrophenyl ATP, with a half-maximal affinity of 2 microM. By both assays competition was observed with ATP or ADP, but not with AMP, GTP, CTP, or UTP. In both nucleotide binding assays, Mg2+ was required, but neither arsenite nor antimonate had any affect. In contrast, the ArsA protein exhibited an ATPase activity which was dependent on the presence of arsenite or antimonate. The results suggest that the ArsA protein is the catalytic subunit of an oxyanion-translocating ATPase.

Lactose permease of Escherichia coli: properties of mutants defective in substrate translocation.

Mutants of lactose permease of Escherichia coli with amino acid changes (Gly-24----Glu; Gly-24----Arg; Pro-28---Ser; Gly-24, Pro-28----Glu-Ser and Gly-24, Pro-28----Arg-Ser) within a putative membrane-spanning alpha-helix (Phe-Gly-Leu-Phe-Phe-Phe-Phe-Tyr-Phe-Phe-Ile-Met-Gly- Ala-Tyr-Phe-Pro-Phe-Phe-Pro-Ile) are incorporated into the cytoplasmic membrane. The mutant proteins retain the ability to bind galactosides, and the affinity for several substrates is actually increased. However, the rate of active transport is decreased to 0.01% of the wild-type rate in the mutants carrying Arg-24 or Arg-24, Ser-28. Kinetic analysis demonstrates that the two mutants require 10 min to cause occupied binding sites for galactoside and H+ to change their exposure from the periplasm to the cytoplasm as compared to 50 ms in the wild type. The effect is less pronounced when these sites are unoccupied.

Does the lactose carrier of Escherichia coli function as a monomer?

The purified lactose carrier of Escherichia coli (product of the lac Y gene) is shown to be a monomer in detergent micelles of dodecyl-O-beta-D-maltoside. The negative-dominant phenotype of mutant carriers (lacY-d mutants) could not be verified by measurements of the rate of galactoside transport in lacY+/Y-d diploid strains. It is proposed that the membrane-embedded carrier functions as a monomer in galactoside-H+ symport.