C1 proteins: a class of high-mobility-group non-histone chromosomal proteins from the fruit fly Ceratitis capitata

1. CM-cellulose chromatography of a fraction soluble in 5% perchloric acid from Ceratitis capitata chromatin yields three proteins, C1a1, C1a2 and C1b, which have been purified to electrophoretical homogeneity. 2. C1a1, C1a2 and C1b analyse like high mobility group (HMG) non-histone chromosomal proteins, although they do not exactly correspond with those from vertebrates. It is proposed that C1 proteins, as well as Drosophila D1 [Rodríguez Alfageme et al. (1980) Chromosoma, 78, 1-31] are representative of a class of insect-specific HMG proteins. Tryptic fingerprints show that C1a1 and C1a2 are very similar, but C1b is a somewhat distinct protein. Circular dichroism studies have shown that these preparations do not appreciably fold on increasing ionic strength. 3. The interactions between DNA and C1 proteins have been studied. These proteins precipitate DNA in 0.15 M NaCl, 0.015 M sodium citrate and the precipitation curves are cooperative. Soluble complexes between C1 proteins and DNA could be prepared in low ionic strength media and their thermal denaturation profiles obtained. C1 proteins do not destabilize DNA under the conditions used to prepare the complexes but the three proteins stabilize DNA to a different degree. From these studies it has been concluded that the association constant of C1b to DNA is smaller than that of C1a1 and C1a2.     

High affinity inhibition of Ca(2+)-dependent K+ channels by cytochrome P-450 inhibitors.

The Ca(2+)-dependent K+ channel of human red cells was inhibited with high affinity by several imidazole antimycotics which are potent inhibitors of cytochrome P-450. IC50 values were (in microM): clotrimazole, 0.05; tioconazole, 0.3; miconazole, 1.5; econazole, 1.8. Inhibition of the channel was also found with other drugs with known cytochrome P-450 inhibitory effect. However, no inhibition was obtained with carbon monoxide (CO). This suggests that, given the high selectivity of the above inhibitors for the heme moiety, a different but closely related to cytochrome P-450 kind of hemoprotein may be involved in the regulation of the red cell Ca(2+)-dependent K+ channel. Clotrimazole also inhibited two other charybdotoxin-sensitive Ca(2+)-dependent K+ channels, those of rat thymocytes (IC50 = 0.1-0.2 microM) and of Ehrlich ascites tumor cells (IC50 = 0.5 microM). Imidazole antimycotics inhibit also receptor-operated Ca2+ channels (Montero, M., Alvarez, J. and García-Sancho, J. (1991) Biochem. J. 277, 73-79). This suggests that both Ca2+ and Ca(2+)-dependent K+ channels might have a similar regulatory mechanism involving a cytochrome.     

Effects of progesterone and estradiol on the proliferation of phytohemagglutinin-stimulated human lymphocytes

In this paper we report on a study to elucidate whether the response of human lymphocytes to mitogenic stimulation was modified by physiological changes which occur during the menstrual cycle. Experiments with untreated cultures showed intra-individual variation to mitogen stimulation in female lymphocyte cultures, but a significant correlation between the menstrual cycle and the proliferation kinetics of lymphocytes was not found. Consequently, we performed experiments in which two of the hormones that regulate the menstrual cycle in women, estradiol and progesterone, were added to cultured human lymphocytes obtained from both men and women. The results indicate that both hormones at physiological concentrations have the capacity to modify the proliferation of PHA-stimulated human lymphocytes. Therefore, both hormones could play a role in the induction of the intra-individual variation observed in the untreated female cultures. However, in vivo other factors could also modify the proliferation kinetics of human lymphocytes preventing the demonstration of the effects of a single factor, such as the hormonal changes occurring during the menstrual cycle.     

Vanadate,alcohol or both increase passive membrane permeability of neuro-2a cells; Lesser sensitivity of Hep-2 cells

Neuro-2a cells incubated for 1 hour with 0.1 mM vanadate showed an increase in cell membrane permeability. This effect is dose dependent, e.g. with 0.01 mM, 0.1 mM and 1 mM vanadate, there was {20, 30 and 40% increase. In contrast, no alteration in permeability was observed in HEp-2 cells under the same conditions.Ethanol (3%, 1 h incubation) also enhanced membrane permeability. The increase was also greater with Neuro-2a cells ({80%) than with HEp-2 cells (～30%). When the cells were incubated with ethanol plus vanadate (0.1 mM), there was a marked potentiation ({200%) in cell membrane permeability in Neuro-2a cells, and again a lesser increase in permeability ({50%) with HEp-2 cells.These results seem to be due to a preferential effect of vanadate on passive permeability of Neuro-2a cells because parallel measurements demonstrate equal inhibition of (Na⁺K) ATPase with both Neuro-2a and HEp-2 cells.     

Chromosome polymorphism in populations of the grasshopper Trimerotropis pallidipennis from southern Argentina

Three populations of the grasshopper Trimerotropis pallidipennis from southern Argentina have been studied cytologically. A very characteristic B-chromosome was found in all three. They also showed geographical variability in respect of the presence of pericentric inversions, and the inversion system was found to influence chiasma frequency. The Laguna Blanca population, which is on the hypothetical pathway the species is believed to have followed during its migration from northern to southern latitudes, has the same karyotype composition as the N. American form, with fixed inversions in the 3 largest autosomes and the X-chromosome. Its members have a high total chiasma frequency and a great number of interstitial chiasmata. The Sierra de la Ventana population, situated at the absolute eastern border of the species distribution is highly polymorphic with respect to the presence of inversions in the medium chromosomes. Its members have the lowest total chiasma frequency and a greatly reduced number of interstitial chiasmata. Situated geographically between the other two, the Choele-Choel population has the highest frequency of inversions and many of them are homozygous. Its members have a higher total chiasma frequency than that observed in specimens from Sierra de la Ventana, and a greatly reduced number of interstitial chiasmata, similar to that observed in individuals from the latter population.     

DNA vaccination can break immunological tolerance to PrP in wild-type mice and attenuates prion disease after intracerebral challenge

Transmissible spongiform encephalopathies (TSEs) can be ameliorated by prion protein (PrP)-specific antibodies, but active immunization is complicated by immune tolerance to the normal cellular host protein (PrP(C)). Here, we show that DNA immunization of wild-type mice can break immune tolerance against the prion protein, resulting in the induction of PrP-specific antibody and T-cell responses. PrP immunogenicity was increased by fusion to the lysosomal targeting signal from LIMPII (lysosomal integral membrane protein type II). Although mice immunized with a PrP-LIMPII DNA vaccine showed a dramatic delay in the onset of early disease signs after intracerebral challenge, immunization against PrP also had some deleterious effects. These results clearly confirm the feasibility of using active immunization to protect against TSEs and, in the absence of effective treatments, indicate a suitable alternative for combating the spread of these diseases.     

Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity

Aryl-hydroxylating dioxygenases are of interest for the degradation of persistant aromatic pollutants, such as polychlorobiphenyls (PCBs), or as catalysts for the functionalization of aromatic scaffolds. In order to achieve dioxygenation of technical mixtures of PCBs, enzymes with broadened or altered substrate ranges are essential. To alter the substrate specificity of the biphenyl dioxygenase (BphA) of Burkholderia xenovorans LB400, we applied a directed evolution approach that used structure-function relationship data to target random mutageneses to specific segments of the enzyme. The limitation of random amino acid (AA) substitutions to regions that are critical for substrate binding and the exclusion of AA exchanges from positions that are essential for catalytic activity yielded enzyme variants of interest at comparatively high frequencies. After only a single mutagenic cycle, 10 beneficial variants were detected in a library of fewer than 1,000 active enzymes. Compared to the parental BphA, they showed between 5- and 200-fold increased turnover of chlorinated biphenyls, with substituent patterns that rendered them largely recalcitrant to attack by BphA-LB400. Determination of their sequences identified AAs that prevent the acceptance of specific PCBs by the wild-type enzyme, such as Pro334 and Phe384. The results suggest prime targets for subsequent cycles of BphA modification. Correlations with a three-dimensional model of the enzyme indicated that most of the exchanges with major influence on substrate turnover do not involve pocket-lining residues and had not been predictable through structural modeling.