Contribution of histones H2A and H2B to the folding of nucleosomal DNA

We have studied the structural properties of nucleosomal particles deficient in histones H2A and H2B produced by modification of histone amino groups with dimethylmaleic anhydride [Jordano, J., Montero, F., & Palacián, E. (1984) Biochemistry (preceding paper in this issue)]. Digestion with DNase I of residual particles containing only 15% of the original H2A . H2B complement produces only discrete DNA fragments no longer than 70 nucleotides. As compared with the original nucleosomes, thermal denaturation of the residual particles shows a decrease from 140 to about 90 in the number of nucleotide base pairs per particle that melt at the highest temperature transition as well as a drop in the temperature of this transition. Circular dichroism spectra of the residual particles give ellipticity values around 275 nm, much higher than those corresponding to the control nucleosomes, which appears to indicate a loss in the compact DNA tertiary structure. When regeneration of the modified amino groups of the residual particles takes place in the presence of the complementary fraction containing histones H2A and H2B, but not in its absence, nucleosomal particles with the structural properties of the original nucleosomes are reconstituted. Therefore, the structural change observed in the residual particles can be assigned to the lack of histones H2A and H2B and not to the modified amino groups of the histones present in the residual particles. The results are consistent with the stabilization by histones H2A and H2B of a DNA length of 50-70 base pairs per nucleosome.     

Involvement of Presynaptic Histamine H3 Receptors in the Modulation of Somatostatin Binding and Its Effects on Adenylyl Cyclase Activity in the Rat Frontoparietal Cortex

Abstract: Thioperamide (2 mg/kg, i.p.), a histamine H₃-receptor antagonist, increased the number of somatostatin (SS) receptors, with no change in the affinity constant, in the rat frontoparietal cortex. This effect was prevented by treatment with ( R )-α-methylhistamine (3.2 mg/kg, i.p.), a histamine H₃-receptor agonist. Thioperamide also induced an increase in SS binding in rats pretreated with mepyramine, a histamine H₁-receptor antagonist, or cimetidine, a histamine H₂-receptor antagonist. Pretreatment with mepyramine plus cimetidine administered simultaneously antagonized the thioperamide effect on SS binding. The increase in the number of SS receptors was accompanied by a greater SS-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase (AC) activity in frontoparietal cortical membranes in the thioperamide group. Furthermore, the functional activity of the guanine nucleotide-binding inhibitory protein (G_i protein) was not altered by thioperamide or ( R )-α-methylhistamine administration in frontoparietal cortical membranes. In rats treated with mepyramine plus thioperamide or cimetidine plus thioperamide, the increase in the number of SS receptors was also accompanied by an increased SS inhibition of AC activity. Thioperamide induced a significant increase in SS-like immunoreactivity content in the frontoparietal cortex. Altogether, these results suggest that frontoparietal cortical histamine may play, at least in part, a role in the regulation of the somatostatinergic system.     

Relaxation of reproductive suppression in non-breeding female naked mole-rats, Heterocephalus glaber

Ninety-four non-reproductive female naked mole-rats, from seven colonies, were studied in terms of vaginal perforation, vaginal smears and urinary concentrations of oestradiol-17β and progesterone in relation to the time of parturition of the breeding female, the queen. The study concentrated mainly on the period from nine days prepartum to 13 days postpartum of 12 births. Sixty-eight percent ( n = 253) of the non-reproductive females had detectable urinary concentrations of oestradiol-17β and many of these had perforated vaginas throughout the study period. These females showed a significantly increased urinary concentration of oestradiol six days prior to parturition of the queen. In females with undetectable concentrations of oestradiol-17β, the proportion with perforated vaginas increased from six days prepartum (54%) to reach a peak on the day of parturition (92%) of the queen. Urinary progesterone-concentrations were 0.7nmol/mmol creatinine at some stage in the study period in 90% of the females and scattered short peaks or spikes were experienced by all these females, but without synchronization between the females in a colony and without any detectable correlation with the time of parturition of the queen. Maximal concentrations in some females were comparable to the values in cycling breeding females during the luteal phase, but were of a much shorter duration than in breeding females. Vaginal smears did not show clear cyclic patterns.     

ε-聚赖氨酸抑菌性能的研究进展

ε-聚赖氨酸(ε-poly-L-lysine,ε-PL)是抑菌谱广泛的天然抑菌剂,由通过α-羧基与ε-氨基连接的25–35个赖氨酸聚合而成。ε-PL主要由白色链霉菌发酵生产所得,比化学生产更加高效和环保。ε-PL具有水溶性好、耐热和对环境无污染等特点,具有良好的应用前景。本文从发酵生产入手,着重综述了ε-PL对各种微生物抑菌性能、抑菌机制及抑菌机制模型的研究进展。推测ε-PL是通过对细胞膜的破坏而改变细胞的通透性,或者作用到细胞内引起活性氧(reactive oxygen species, ROS)胁迫而影响调节基因的表达,从而起到抑菌作用。根据这2种抑菌方式分别建立了相应的抑菌模型,即毡毯模型和ROS诱导细胞凋亡模型。本文可为ε-PL对微生物抑制性能的深入研究提供依据,同时也提出了ε-PL抑菌机制的新模型,为扩展ε-PL应用领域提供了一定的参考。     

Effects of temperature and photoperiod on flowering in Hyptis brevipes

Few tropical species have been tested for their flowering response under controlled conditions. Hyptis brevipes Poit, is an annual herb, commonly found in wet margins of streams and ponds, being considered a weed for some perennial plantations in Brazil. Under experimental glasshouse conditions, this species proved to be an obligate short-day plant. Flowering was delayed when photoperiods longer than 8 h were given, the critical photoperiod being between 12 and 13 h. When both temperature and photoperiod were controlled, at 20°C a longer protoperiod (by almost 1 h) is still inductive compared to 25 and 30°C. The number of short-day cycles required for full induction is relatively high and dependent upon temperature; at 20°C or above, 10 cycles are adequate, but at 15°C, more short-day cycles are needed. The number of inflorescences formed as well as the floral index vary according to daylength × temperature × inductive cycle number, allowing flowering to be assessed quantitatively. Long days are inhibitory to flowering, either suppressing it completely (when symmetrically intercalated among 24 inductive cycles) or preventing the floral index from increasing.     

Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.

Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.     

Dissociation of single-stranded DNA from nucleosomes following modification with acetic anhydride

Modification with acetic anhydride of nucleosomes from chicken erythrocytes at low ionic strength (less than 0.1 M NaCl) is accompanied by the formation of residual particles and the release of free DNA. This DNA has been identified as single-stranded by thermal denaturation, digestion with nuclease S1, and elution from hydroxyapatite. In contrast, if modification takes place at 0.6 M NaCl, the liberated DNA is mainly double-stranded. The release of the free energy stored in folded nucleosomal DNA, triggered by the weakening of lysine-DNA interactions which takes place upon modification, might be responsible for the observed denaturation of DNA at low ionic strength.     

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.     

Sunlight and the survival of enteric bacteria in natural waters

Escherichia coli and some salmonellas were exposed in seawater and freshwater to natural sunlight, visible light of comparable intensity, and light containing a similar proportion of u.v. as natural sunlight but of a much lower intensity. Direct viable bacterial counts and culturable counts on selective and non-selective media were made at intervals. The rate of decrease in numbers of culturable bacteria was significantly faster in seawater than in freshwater when exposed to natural sunlight. No significant difference was found between the rates of decrease in numbers of culturable bacteria in seawater and those in freshwater when bacteria were exposed to light with a small u.v. component of similar intensity. The effect of salinity on loss of culturability is, therefore, more significant in the presence of u.v. radiation. Direct counts by the acridine orange direct viable count method decreased much more slowly than the culturable counts in seawater but comparably with culturable counts in freshwater in natural sunlight. Direct viable counts and culturable counts decreased at a similar rate in seawater and in freshwater in visible light. This may signify the evolution of enteric bacteria towards a viable but non-culturable form in seawater when exposed to natural sunlight. The presence of humic acids significantly reduced loss of culturability but only in low salinity conditions. Salinity appears to be an important factor influencing culturability in bacteria exposed to sunlight.     

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.