The structure of nucleosome core particles as revealed by difference Raman spectroscopy.

Raman spectra have been observed of nucleosome core particles (I) prepared from chicken erythrocyte chromatin, its isolated 146 bp DNA (II), and its isolated histone octamer (H2A+H2B+H3+H4)2 (III). By examining the difference Raman spectra, (I)-(II), (I)-(III), and (I)-(II)-(III), several pieces of information have been obtained on the conformation of the DNA moiety, the conformation of the histone moiety, and the DNA-histone interaction in the nucleosome core particles. In the nucleosome core particles, about 15 bp (A.T rich) portions of the whole 146 bp DNA are considered to take an A-form conformation. These are considered to correspond to its bent portions which appear at intervals of 10 bp.     

Indomethacin, but not dazoxiben, reduced lung fluid filtration after E. coli infusion

Goats were divided into three groups and given infusions of live Escherichia coli bacteria. Group I received no treatment, group II was treated with indomethacin (a cyclooxygenase inhibitor), and group III with dazoxiben (a thromboxane synthase inhibitor). Double indicator-dilution extravascular lung water (EVLW) in group I was significantly different from the treated groups. There was an early increase in EVLW in group I and group III but not in group II animals. At 6 h EVLW's in group I, group II, and group III were 100, 45, and 30% above base line, respectively. Lymph flow (QL) and lymph-to-plasma protein ratio (L/P) was not statistically different between groups. Estimated total fluid filtration [QL + d(EVLW)/dt] in group I and III was markedly elevated between 0 and 1.5-2 h after E. coli infusion. Cardiac output (QT) decreased to 40% of base line in group I, and it decreased slightly in group II because of the indomethacin but did not decrease after E. coli. QT decreased in group III but recovered more rapidly than group I. Mean pulmonary arterial pressure increased more rapidly in group I and reached a higher peak than either treated group. At 6 h these groups had similar pulmonary arterial and pulmonary arterial wedge pressures. We conclude that 1) indomethacin but not dazoxiben blocks the early increase in total fluid filtration after bacterial infusion, 2) dazoxiben does not prevent the increased endothelial permeability resulting from infusion of live bacteria, and 3) indomethacin may somewhat ameliorate the endothelial permeability change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parameters of dietary selenium and vitamin E deficiency in growing rabbits.

4 x 5 growing female rabbits (New Zealand White) with an initial live weight of 610 +/- 62 g were fed a torula yeast based semisynthetic diet low in selenium (<0.03 mg/kg diet) and containing <2 mg alpha-tocopherol per kg (group I). Group II received a vitamin E supplementation of 150 mg alpha-tocopherylacetate per kg diet, whereas for group III 0.40 mg Se as Na-selenite and for group IV both supplements were added. Selenium status and parameters of tissue damage were analyzed after 10 weeks on experiment (live weight 2,355 +/- 145 g). Selenium depletion of the Se deficient rabbits (groups I and II) was indicated by a significantly lower plasma Se content (group I: 38.3 +/- 6.23 microg Se/mL plasma, group II: 42.6 +/- 9.77, group III: 149 +/- 33.4, group IV: 126 +/- 6.45) and a significantly lower liver Se content (group I: 89.4 +/- 18.2 microg/kg fresh matter, group II: 111 +/- 26.2) as compared to the Se supplemented groups III (983 +/- 204) and IV (926 +/- 73.9). After 5 weeks on the experimental diets differences in the development of plasma glutathione peroxidase were observed. As compared to the initial status group (45.2 +/- 4.50) pGPx activity in mU/mg protein was decreased in group I (19.1 +/- 7.08), remained almost stable in the vitamin E supplemented group II (46.3 +/- 11.2) whereas an elevated enzyme activity was measured in the Se supplemented groups III (62.4 +/- 23.9) and IV (106 +/- 19.9). In the rabbit organs investigated 10 weeks of Se deficiency caused a significant loss of Se dependent cellular glutathione peroxidase activity (GPx1) of 94% (liver), 80% (kidney), 50% (heart muscle) and 60% (musculus longissimus dorsi) in comparison to Se supplemented control animals. Damage of cellular lipids and proteins in the liver was due to either Se or vitamin E deficiency. However damage was most severe under conditions of a combined Se and vitamin E deficiency. It can be concluded that the activity of plasma glutathione peroxidase is a sensitive indicator of Se deficiency in rabbits. The loss of GPx1 activity indicates the selenium depletion in various rabbit organs. Both selenium and vitamin E are essential and highly efficient antioxidants which protect rabbits against lipid and protein oxidation.     

Negative effect of a cis-acting pBR322 element on adenovirus E1a gene expression

A sequence element within plasmid pBR322 has a cis-acting negative effect on the expression of a cloned Ad gene in transient expression assays. The negative element is located between the PvuII and Tth111I restriction sites on pBR322 (nt 2068-2223). This element was also shown to be responsible for the decrease in focus number, when plasmids carrying Ad2 E1 genes were used to transform baby rat kidney cells. In a similar manner, this element diminished the number of G418-resistant cell foci, when plasmids containing the neo marker gene under the control of E1a promoter were used. Plasmid stability in transfected cells was not modified by deletion of this cis-acting negative element.     

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D [1H,15N] HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation ( K(D)) and off-rate ( k(off)) constants at 30 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a k(off) of 5000 s(-1) for site II Ca2+ dissociation at 30 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), and cTnI(147-163), were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K(D) and k(off) values are 1 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI(128-147) and cTnI(147-163) from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI(34-71) from cTnC may be too slow for this process.     

The beta globin gene cluster of the prosimian primate Galago crassicaudatus: nucleotide sequence determination of the 41-kb cluster and comparative sequence analyses.

The nucleotide sequence of the beta globin gene cluster of the prosimian Galago crassicaudatus has been determined. A total sequence spanning 41,101 bp contains and links together previously published sequences of the five galago beta-like globin genes (5'-epsilon-gamma-psi eta-delta-beta-3'). A computer-aided search for middle interspersed repetitive sequences identified 10 LINE (L1) elements, including a 5' truncated repeat that is orthologous to the full-length L1 element found in the human epsilon-gamma intergenic region. SINE elements that were identified included one Alu type I repeat, four Alu type II repeats, and two methionine tRNA-derived Monomer (type III) elements. Alu type II and Monomer sequences are unique to the galago genome. Structural analyses of the cluster sequence reveals that it is relatively A+T rich (about 62%) and regions with high G+C content are associated primarily with globin coding regions. Comparative analyses with the beta globin cluster sequences of human, rabbit, and mouse reveal extensive sequence homologies in their genic regions, but only human, galago, and rabbit sequences share extensive intergenic sequence homologies. Divergence analyses of aligned intergenic and flanking sequences from orthologous human, galago, and rabbit sequences show a gradation in the rate of nucleotide sequence evolution along the cluster where sequences 5' of the epsilon globin gene region show the least sequence divergence and sequences just 5' of the beta globin gene region show the greatest sequence divergence.