Imidazole: A selective inhibitor of thromboxane synthetase

Imidazole inhibits the enzymatic conversion of the endoperoxides (PGG₂ and PGH₂) to thromboxane A₂ by platelet microsomes (IC₅₀: 22 μg/ml; determined by bioassay). The inhibitor is selective, for prostaglandin cyclo-oxygenase is only affected at high doses. Radiochemical data confirms that imidazole blocks the formation of ¹⁴C-thromboxane B₂ from ¹⁴C-PHG₂. Several imidazole analogues and other substances were tested but only 1-methyl-imidadole was more potent that imidazole iteself. The use of imidazole of inhibit thromboxane formation could help to elucidate the role of thromboxanes in physiology or pathophysiology.     

A study of the dynamic properties of actomyosin systems by quasi-elastic light scattering

A laser light source and a digital autocorrelator were employed in the study of the molecular dynamics of acto-heavy meromyosin during the splitting of ATP. Low protein concentrations were used, so that molecular and not gel properties were evident. The addition of Mg²⁺ to acto-heavy meromyosin solutions in the presence of ATP caused a marked widening of the spectrum at high scattering angles. No such change was observed when chemically inactivated heavy meromyosin was used, when actin was cross-linked or when the proteins were in a high ionic strength solution. The data can be interpreted in terms of pronounced change in flexibility of acto-heavy meromyosin induced by active mechanochemical coupling.     

Nanosecond decay studies of a fluorescence probe bound to apomyoglobin.

Excited state interactions of N-(p-tolyl)-2-aminonaphthalene-6-sulfonate (2, 6 p-TNS) bound to apomyoglobin were studied by nanosecond time-resolved emission spectroscopy. A dynamic interaction of the excited dye molecule with its binding site, associated with a significant change in the emission energy with time, was observed. The decay kinetics were found to be complex and consistent with the kinetic model for solvent relaxation as proposed by Bakhshiev et al. (Opt. Spectrosc. 21:307. 1966). The behavior of 2, 6 p-TNS bound to apomyoglobin was found to be qualitatively similar to that of the dye dissolved in a viscous solvent such as glycerol or adsorbed to egg lecithin vesicles. The detailed information obtained by following the changes in emission spectra of fluorescent probes on the nanosecond time scale leads to a better understanding of their interactions with biological systems.     

Cloning and characterization of the tomato karyopherin alpha1 gene promoter

The karyopherin alpha1 (LeKAPalpha 1) gene of tomato (Lycopersicon esculentum) encodes a receptor involved in nuclear import. To analyze the expression pattern of this gene, a genomic clone containing its upstream region was isolated and sequenced. To study the promoter functionality, a 2170 bp fragment (LM1), was fused to glucuronidase (GUS) and introduced into petunia cells by particle bombardment. For further characterization of the promoter, one inverse and three deletion constructs were studied in cell suspension. To follow its expression in tobacco leaves, transgenic plants expressing GUS under the control of the LM1 promoter were made. Expression of LM1-GUS was largely restricted to actively growing leaf regions, suggesting possible involvement of active cell division and plant growth regulators in LeKAPalpha 1 expression.     

Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine (polyQ) tract expansion near the N terminus of huntingtin (Htt). Proteolytic processing of mutant Htt and abnormal calcium signaling may play a critical role in disease progression and pathogenesis. Recent work indicates that calpains may participate in the increased and/or altered patterns of Htt proteolysis leading to the selective toxicity observed in HD striatum. Here, we identify two calpain cleavage sites in Htt and show that mutation of these sites renders the polyQ expanded Htt less susceptible to proteolysis and aggregation, resulting in decreased toxicity in an in vitro cell culture model. In addition, we found that calpain- and caspase-derived Htt fragments preferentially accumulate in the nucleus without the requirement of further cleavage into smaller fragments. Calpain family members, calpain-1, -5, -7, and -10, have increased levels or are activated in HD tissue culture and transgenic mouse models, suggesting they may play a key role in Htt proteolysis and disease pathology. Interestingly, calpain-1, -5, -7, and -10 localize to the cytoplasm and the nucleus, whereas the activated forms of calpain-7 and -10 are found only in the nucleus. These results support the role of calpain-derived Htt fragmentation in HD and suggest that aberrant activation of calpains may play a role in HD pathogenesis.     

Protection of phosphoglycerate kinase against in vitro aging by selective cysteine methylation

Recent studies have demonstrated that the aging effects in phosphoglycerate kinase (PGK) may be simulated in vitro by prolonged incubation of the enzyme under nonreducing conditions followed by reduction with excess 2-mercaptoethanol. The simulated-old enzyme thus produced appears to be identical to native old PGK and, like the latter enzyme, may be successfully rejuvenated by an unfolding-refolding procedure. A model for PGK aging was proposed in which initial and reversible oxidation of the enzyme is followed by conformational modifications that persist after the enzyme is re-reduced. The role of specific cysteine oxidation in the initial step of PGK aging was tested in the present study by selectively methylating the fast-reacting cysteine residues in this enzyme, thus blocking the putative oxidation sites, and producing in vitro a young form of PGK that is immune to aging. The methylation was performed by treating the enzyme with excess iodomethane and monitoring the reaction by determining the concentration of unreacted cysteines in the enzyme as a function of time. Unmethylated controls were incubated similarly but in the absence of iodomethane. The methylated as well as control samples of PGK were subsequently incubated under conditions which caused native young PGK to develop the age-related effects and become identical to native old PGK. In contrast, the methylated enzyme remained identical to young PGK. These findings strongly support the hypothesis that cysteine oxidation is an essential step in the aging of rat muscle phosphoglycerate kinase.     

Noxious stimulation of animals by brief intense laser induced heat: Advantages to pharmacological testing of analgesics

A new technique for experimental pain inducing in animals is described. The technique, which involves brief and intense pulses of heat generated by a CO₂ laser, has several advantages over other procedures of noxious thermal stimulation. The animals do not have to be restrained, they do not adapt to the stimulation, and they respond by a rich behavioral repertoire which is related to the stimulus intensity. The usefulness of the technique in pharmacological testing of analgesics is illustrated in a preliminary study demosntrating a remarkable elevation of the thresholds to this type of noxious thermal stimulation after subcutaneous morphine injections.     

Ferredoxin from Halobacterium of the Dead Sea. Structural properties revealed by fluorescence techniques

The two tryptophan residues of ferredoxin from Halobacterium of the Dead Sea differ in their fluorescence characteristics. One of these tryptophan residues (class 1) absorbs more to the red and is thus probably in a more apolar environment than the other (class 2). Upon removal of the ferric ions, i.e., in the apoferredoxin, a 2.2-fold increase in the quantum yield of fluorescence is observed. A double exponential decay of the fluorescence is found for ferredoxin, reduced ferredoxin, as well as for the apoferredoxin. The longer decay time assumes a constant value of 6.9 ns in all three cases, indicating that it originates in a tryptophan residue which is not affected by changes in the Fe³⁺ binding site (class 2 tryptophan). The shorter decay component increases gradually from 0.55 ns in oxidized ferredoxin, through 0.80 ns in the reduced ferredoxin to 1.24 ns in the apoprotein. This decay component is thus assumed to be largely due to the second tryptophan residue of the protein (class 1) located close to the Fe³⁺ binding site. On the other hand, the relative decay amplitude of the class 2 tryptophan is doubled upon formation of apoferredoxin. It is concluded that the class 1 tryptophan is quenched by the active site ferric ions and that the class 2 tryptophan is partially exposed to a polar environment. Whereas class 1 tryptophan may be similar to the single nonfluorescent tryptophan of spinach ferredoxin, class 2 tryptophan is found in a peptide which is present only in halophilic ferredoxins. Conformational changes occur in the molecule upon removal—but not reduction—of the ferric ions, causing the environment of the class 2 tryptophan to become more hydrophobic. It is possible that the class 1 tryptophan is associated with the occurrence of a higher redox potential in this ferredoxin, when compared with chloroplast-type ferredoxins.