The use of 2-hydroperoxytetrahydrofuran as a reagent to sequence cytosine and to probe non-Watson-Crick DNA structures.

2-Hydroperoxytetrahydrofuran (THF-OOH) can be employed to sequence cytosine (C) and to probe for non-canonical DNA structures involving C. Using 32P-labeled oligomers and a DNA restriction fragment, it is demonstrated that THF-OOH has a strong preference for Cs in single-stranded (s-s) DNA regions, and in bulges, loops and mismatches. The reactivity of C is diminished below pH 6.0, but is not affected by substitution of 5-methylcytosine. To demonstrate the utility of the reagent, it is directly compared to methoxylamine and chloroacetaldehyde, two other reagents commonly used to chemically probe C residues in non-Watson-Crick DNA structures.     

Glyoxylate transamination in intact leaf peroxisomes

Intact spinach (Spinacia oleracea L.) leaf peroxisomes were supplied with glycolate and one to three of the amino acids serine, glutamate, and alanine, and the amount of the respective α-keto acids formed in glyoxylate transamination was assayed. At 1 millimolar glycolate and 1 millimolar each of the three amino acids in combination, the transamination reaction reached saturation; reduction of either glycolate or amino acid concentration decreased the activity. The relative serine, glutamate, and alanine transamination at equal amino acid concentrations was roughly 40, 30, and 30%, respectively. The three amino acids exhibited mutual inhibition to one another in transamination due to the competition for the supply of glyoxylate. In addition to this competition for glyoxylate, competitive inhibition at the active site of enzymes occurred between glutamate and alanine, but not between serine and glutamate or alanine. Alteration of the relative concentrations of the three amino acids changed their relative transamination. Similar work was performed with intact oat (Avena sativa L.) leaf peroxisomes. At 1 millimolar of each of the three amino acids in combination, the relative serine, glutamate, and alanine transamination was roughly 60, 23, and 17%, respectively. Similarly, alteration of the relative concentration of the three amino acids changed their relative transamination. The contents of the three amino acids in leaf extracts were analyzed, and the relative contribution of the three amino acids in glycine production in photorespiration was assessed and discussed.     

Murine leukemia cell hybrids: The quantity of TL antigens expressed by parental and hybrid cells fails to correlate with their sensitivity to TL antibody and complement

The quantity of thymus-leukemia (TL) antigens expressed by murine leukemia cells is significantly greater than that expressed by somatic hybrids of such cells. Based upon the results of ¹²⁵I-lactoperoxidase labeling and antibody absorption procedures, and corrected for size differences between the two cell types, the quantity of TL antigens expressed by RADA-1 cells, a radiation-induced murine leukemia cell line of strain A/J mice, is approximately 5.0 times greater than that of somatic hybrids of RADA-1 and LM(TK)^? cells. LM(TK)^? cells are a thymidine kinase-deficient TL(-) mouse fibroblast cell line. The quantity of TL antigens expressed is related only in part to their susceptibility to lysis by TL antibodies and guinea pig complement (GPC). RADA-1 cells resist lysis. The quantity of TL antigens expressed by RADA-1 cells is analogous to that formed by nonneoplastic thymocytes obtained from F₁ hybrids of two strains of TL(+) and TL(-) mice; cells from both strains are sensitive to TL antiserum and GPC. ASL-1 cells, a spontaneously occurring leukemia cell line of A/J mice, express TL antigens in significantly higher quantities than any of the cell types examined. Exposed to TL antisera, the quantity of TL antigens of ASL-1 cells, but not that of hybrid cells, gradually diminishes. ASL-1 cells convert over a 6-h period of exposure to antibody and guinea pig complement (GPC) resistance; hybrid cells remain sensitive. However, ASL-1 cells converted to TL antibody and GPC resistance continue for a time to express TL antigens in quantities similar to that of sensitive F₁ thymocytes and resistant RADA-1 cells. RADA-1 X LM(TK)^? hybrid cells, which are sensitive to TL antibodies and GPC, express the lowest quantities of TL antigens of any of the cell types examined. It is likely that differences in the quantities of TL antigens expressed by different cell lines reflect genetic mechanisms controlling TL antigen expression. The failure of TL antisera to affect the quantities of TL antigens expressed by hybrid cells is taken as an indication that genetic controls governing antigen expression may be distinguished from those involved in regulating responsiveness to specific antiserum.     

Micromanipulation of mitotic chromosomes in PTK2 cells using laser-induced optical forces ("optical tweezers")

To study the potential use of optical forces to manipulate chromosome movement, we have used a Nd:YAG laser at a wavelength of 1.06 microns focused into a phase contrast microscope. Metaphase and anaphase chromosomes were exposed while being monitored by video microscopy. The results indicated that when optical forces were applied to late-moving metaphase chromosomes on the side closest to the nearest spindle pole, the trapped chromosomes initiated movement to the metaphase plate. The chromosome velocities were two to eight times the normal rate depending on the chromosome size, geometry, and trapping site. At the initiation of anaphase, a pair of chromatids could be held by the optical trap and kept motionless throughout anaphase while the other pairs of chromatids separated and moved to opposite spindle poles. As a result, the trapped chromosome either was incorporated into one of the daughter cells or was lost in the cleavage furrow, or the two chromatids eventually separated and moved to their respective daughter cells. If the trap was removed at the beginning of anaphase B, the chromosome moved back to the poles. Our experiments demonstrate that the laser-induced optical force trap is a potential new technique to study noninvasively the mitotic spindle of living cells.     

七坪林场常绿阔叶林凋落物研究初报

凋落物即来自林冠的落叶、落枝和落花果等有机残体。既是林木自身的代谢产物,又是森林土壤养分的重要来源,在森林生态系统养分循环中发挥重要的作用。有关天然林凋落物的研究,国内已有一些     

显微高速摄影技术及其在血液微流变学研究中的应用

本文选用金黄地鼠的微血管,利用显微高速摄影技术,放大微观流场和血细胞,连续地“冻结”短瞬间的变化状态,把物理图象呈现在胶片上,经图像分析和数据处理,从定性及定量方面研究血液微流变学,为生命科学和医学研究提供了又一种新的方法。     

Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli.

The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.     

Variations in the turn-forming characteristics of N-acyl proline units.

We have examined intramolecular hydrogen bonding in four homologous compounds, N-acetyl-, N-propionyl-, N-i-butyryl-, and N-pivaloyl-proline-methylamide, in methylene chloride, by means of 1H-nmr and ir measurements. At room temperature, the major trans conformer of MeCO-Pro-NHMe appears to be approximately 68% intramolecularly hydrogen bonded, the trans conformers of EtCO-Pro-NHMe and i-PrCO-Pro-NHMe are approximately 75% intramolecularly hydrogen bonded, and t-BuCO-Pro-NHMe is approximately 50% intramolecularly hydrogen bonded. Thus, the internally hydrogen-bonded state (C7 or gamma-turn) is significantly less populated for the N-pivaloyl compound than for the other three molecules in this series. Variable temperature measurements indicate that for each proline derivative there is very little enthalpic difference between the intramolecularly hydrogen-bonded and nonhydrogen bonded states of the trans rotamer. Changing the N-terminal acyl group also affects intramolecular hydrogen bonding (including beta-turn formation) in end-blocked Pro-Gly dipeptides.     

M?ssbauer study of the native, reduced and substrate-reacted Desulfovibrio gigas aldehyde oxido-reductase.

The Desulfovibrio gigas aldehyde-oxido-reductase contains molybdenum and iron-sulfur clusters. M?ssbauer spectroscopy was used to characterize the iron-sulfur clusters. Spectra of the enzyme in its oxidized, partially reduced and benzaldehyde-reacted states were recorded at different temperatures and applied magnetic fields. All the iron atoms in D. gigas aldehyde oxido-reductase are organized as [2Fe-2S] clusters. In the oxidized enzyme, the clusters are diamagnetic and exhibit a single quadrupole doublet with parameters (delta EQ = 0.62 +/- 0.02 mm/s and delta = 0.27 +/- 0.01 mm/s) typical for the [2Fe-2S]2+ state. M?ssbauer spectra of the reduced clusters also show the characteristics of a [2Fe-2S]1+ cluster and can be explained by a spin-coupling model proposed for the [2Fe-2S] cluster where a high-spin ferrous ion (S = 2) is antiferromagnetically coupled to a high-spin ferric ion (S = 5/2) to form a S = 1/2 system. Two ferrous sites with different delta EQ values (3.42 mm/s and 2.93 mm/s at 85 K) are observed for the reduced enzyme, indicating the presence of two types of [2Fe-2S] clusters in the D. gigas enzyme. Taking this observation together with the re-evaluated value of iron content (3.5 +/- 0.1 Fe/molecule), it is concluded that, similar to other Mo-hydroxylases, the D. gigas aldehyde oxido-reductase also contains two spectroscopically distinguishable [2Fe-2S] clusters.