Permanent Preservation of Whole Alizarin Red S Skeletons by Clearing and Embedding in Polyester Resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Effects of dicyclohexane derivatives on androgen metabolism in the rat prostate

Dicyclohexane derivatives, which inhibit the binding of testosterone and dihydrotestosterone (DHT) to the androgen-binding protein (ABP) of rat epididymis without interfering with their binding to the androgen receptor, show a similar selectivity in their effects on androgen metabolism. Their ability to inhibit the aromatization of testosterone has been reported previously. This paper demonstrates that they are potent inhibitors of 3 alpha(beta)-hydroxysteroid:NAD(P)+ oxidoreductase activity (3-HSD) in the particulate fraction from rat prostate gland; the values of Ki for their inhibition of this enzyme are similar to that of the Km for DHT as substrate. The dicyclohexane derivatives are markedly less effective against the cytosolic NADPH-dependent 3-HSD, and they do not appear to inhibit testosterone 5 alpha-reductase activity. These characteristics are likely to complicate the proposed use of the dicyclohexane derivatives as probes for the role of ABP in vivo. However, they may be of interest in the study of structure-activity relationships in androgen-metabolizing enzymes, particularly in the examination of the different forms of 3-HSD.     

Radioimmunoassay of ochratoxin A in barley

A simple and rapid radioimmunoassay was developed for the quantitative determination of ochratoxin A in barley. [14C]ochratoxin A, with a specific activity of 130 Ci/mol, was used as the tracer. Toxin levels below 100 ng/ml required a cleanup step. Three methods (the Association of Official Analytical Chemists cleanup method, the solvent partition method, and the Extrelut 3 column cleanup method) were compared.     

Permanent preservation of whole alizarin red S skeletons by clearing and embedding in polyester resins

A one-step clearing and embedding procedure for alizarin red S stained skeletons is described. Embryos are fixed in formalin, skinned and eviscerated. After staining in a 10 mg/liter solution of alizarin red S in 5% aqueous KOH, specimens are dehydrated in a graded series of acetone-polyester monomer solutions. Finally, the specimens are embedded at room temperature in the polyester resin. A special reusable metallic mold is described for embedment of large fetuses. Specimens previously cleared in glycerol can be processed with this method.     

Resonance Raman spectra of the heme in leghemoglobin. Evidence for the absence of ruffling and the influence of the vinyl groups

Resonance Raman spectra of deoxy and carbonmonoxy leghemoglobin (Lb) are compared to the corresponding forms of human adult hemoglobin (HbA). It is found that the heme "core size" indicator line has nearly the same frequency for the two deoxyhemoglobins and the pi-electron density-sensitive line also falls at the same frequency. However, several other modes occur at very different frequencies in the spectra of the two proteins. From an examination of the spectrum of an HbA derivative in which the beta-carbon atoms of the heme vinyl groups were deuterated, it appears that the major differences between deoxy-HbA and -Lb may result from conformational changes in the vinyl groups. No evidence for the suggested ruffling (Irwin, M. J., Armstrong, R. S., and Wright, P. E. (1981) FEBS Lett. 133, 239-243) in deoxy-Lb was found. The spectra of carbonmonoxy-Lb and -HbA were also found to be very different. As in the deoxy case, some of these frequency differences could be attributed to vinyl group conformational differences. However, from the large difference in the pi-electron density-sensitive line, it appears that the vinyl pi-conjugation into the porphyrin in Lb(CO) may be different than it is in HbA(CO). The vinyl conformational differences may be a consequence of the looser heme pocket in Lb than in HbA. The difference in pi-conjugation could make a significant contribution to the difference in ligand binding affinity for these two globins.     

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene.

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.     

Structural characterization of cytochrome c peroxidase by resonance Raman scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.