The effect of carbon dioxide flow rate on the euthanasia of laboratory mice.

Laboratory rodents are commonly euthanized by exposure to gradually increasing concentrations of carbon dioxide (CO2). Current recommended flow rates range between 10 and 30% chamber vol/min and result in insensibility before exposure to painful concentrations (<40%). However, this method causes dyspnea, indicated by deep, rapid breathing. In humans dyspnea is associated with a negative affective experience. Sensations of dyspnea may explain why rodents find CO2 concentrations >3% aversive. This study aimed to assess the effect of CO2 flow rates on time between the onset of dyspnea and various measures of insensibility (recumbency, loss of the righting reflex and loss of the pedal withdrawal reflex) to identify flow rates that minimize the potential experience of dyspnea. The results of this study indicate that a flow rate of 50% chamber vol/min, while holding the CO2 cage concentration just below 40%, minimizes the interval between the onset of labored breathing and recumbency. Using a 50% flow rate this interval averaged (± SE) 30.3 ± 2.9 s versus 49.7 ± 2.9 s at 20% chamber vol/min (F3,22 = 7.83, P = 0.0013). Similarly, the interval between the onset of labored breathing and loss of the righting reflex averaged 38.2 ± 2.4 s at a flow rate of 50% versus 59.2 ± 2.4 s at 20% chamber vol/min of CO2 (F3,22 = 13.62, P < 0.0001). We conclude that higher flow rates reduce the duration of dyspnea, but even at the highest flow rate mice experience more than 30 s between the onset of dyspnea and the most conservative estimate of insensibility.

Impact of Medicaid resources on core public health responsibilities of local health departments in Illinois.

With Illinois' plan to embark on a statewide Medicaid managed care program, the impact of Medicaid resources on core public health responsibilities of local health departments (LHDs) was assessed and found to be substantial. A reduction of $330,000 in core public health activities would likely accompany each $1 million in Medicaid resources lost by these LHDs. Only by actively participating in the planning and implementation of these conversions can public health agencies maintain high productivity and efficiency in addressing core public health responsibilities in their communities.

Design, synthesis and structure of a zinc finger with an artificial beta-turn.

We have incorporated a bicyclic beta-turn mimetic (BTD; beta-turn dipeptide) into a zinc finger, creating a zinc finger with an artificial beta-turn. The designed peptide chelates zinc and has the same fold as the unmodified native zinc finger (finger 3 of the human YY1 protein). A combination of 1H NMR and structure calculations reveals that, in solution, this zinc finger has a fold similar to the known wild-type crystal structure and to other zinc fingers containing the consensus sequence X3-Cys-X4-Cys-X12-His-X3-His-X. The peptide was designed with BTD between the chelating cysteine residues, with BTD forming a type II' beta-turn linking the two strands of a distorted anti-parallel beta-sheet. The C-terminal portion of the peptide forms a helix with zinc co-ordinating histidine residues on successive turns of the helix. This work represents a step towards developing methods by which parts of a target protein may be replaced by peptide mimetics.

Solution structure of a biologically active cyclic LDV peptide analogue containing a type II' beta-turn mimetic.

The solution structure of cyclo-[Gly-Leu-Asp-Val-BTD] (BTD = beta-turn dipeptide) has been determined by two-dimensional 1H-NMR (nuclear magnetic resonance) spectroscopy and systematic conformational searching combined with molecular dynamics studies. The structure contains two hydrogen bonds between the Gly and Val residues, and a type I beta-turn with Leu and Asp at the (i + 1) and (i + 2) positions of the turn. The cyclic compound shows activity in a scintillation proximity assay (SPA) for the inhibition of the interaction between the integrin alpha 4 beta 1 and vascular cell adhesion molecule-1 (VCAM-I). The structure-activity relationship of the LDV sequence is discussed.

Towards meeting the Paracelsus Challenge: The design, synthesis, and characterization of paracelsin-43, an alpha-helical protein with over 50% sequence identity to an all-beta protein.

In response to the Paracelsus Challenge (Rose and Creamer, Proteins, 19:1-3, 1994), we present here the design, synthesis, and characterization of a helical protein, whose sequence is 50% identical to that of an all-beta protein. The new sequence was derived by applying an inverse protein folding approach, in which the sequence was optimized to "fit" the new helical structure, but constrained to retain 50% of the original amino acid residues. The program utilizes a genetic algorithm to optimize the sequence, together with empirical potentials of mean force to evaluate the sequence-structure compatibility. Although the designed sequence has little ordered (secondary) structure in water, circular dichroism and nuclear magnetic resonance data show clear evidence for significant helical content in water/ethylene glycol and in water/methanol mixtures at low temperatures, as well as melting behavior indicative of cooperative folding. We believe that this represents a significant step toward meeting the Paracelsus Challenge.

Stereospecific assignments of the leucine methyl resonances in the 1H NMR spectrum of Lactobacillus casei dihydrofolate reductase.

A general method is described for the stereospecific assignment of methyl resonances in protein NMR spectra based on selective deuteration procedures. A selectively deuterated dihydrofolate reductase from L. casei was prepared by incorporating stereoselectively deuterated L-leucine, (2S,4R)[5,5,5-2H3]leucine. By comparing the COSY spectra of the dihydrofolate reductase-methotrexate complexes formed using deuterated and non-deuterated enzyme the stereospecific assignments for resonances of all 13 leucine residues were obtained by noting the absence of cross-peaks in spectra from the deuterated proteins.