Characterization and Cloning of ODAP Degrading Gene from a Soil Microbe

The strain BYT-1, capable of utilizing ODAP/DAP as a sole source of nitrogen and carbon was identified as Psuedomonas stutzeri by various microbial and biochemical tests. Transformation experiments showed that the ODAP utilizing property Is encoded by the plasmid. Restriction of plasmid DNA with Pstl, followed by cloning of fragments and screening of ODAP containing medium, led to the isolation of a clone with insert size of ?3.3 kb, which encoded ODAP metabolizing property. The growth and ODAP/DAP utilization by this clone (T_B) was almost similar to that of the wild type strain.     

Repair rate in human fibroblasts measured by thymine dimer excorporation

Summary The UV photoproduct, thymine dimer ( ), is excorporated with a remarkably low rate from the DNA of human fibroblasts grown in cell culture. An UV dose of 18 J/m² creates 0.045% (related to thymine). Within the first two days of repair logarithmically growing and quiescent fibroblasts exhibit the same repair rates; thereafter, the proportion of is lower in growing cells due to recovery of DNA replication. Only about 50% of the lesions are excised within 24 h. In quiescent cells, 13% of the thymine dimers originally present can be detected as late as a week after UV-irradiation. Two distinct first-order rate constants indicate that approximately half of the dimers are less accessible to repair. Repair measured by the nucleoid decondensation technique corresponds to the faster repair rate, whereas the slow repair rate cannot be detected by this method. Saturation of repair is found beyond 27 J/m². The remarkably slow rate of excision indicates that thymine dimers are not lethal lesions in human fibroblasts.     

Spectrum of chloroperoxidase compound I

When compound I of chloroperoxidase is formed from the native enzyme the absorption peak in the Soret region diminishes in intensity, and shifts to a maximum absorbance at 367 nm. This unusual Soret spectrum decreases in intensity in a linear fashion as the wavelength increases. The first visible spectrum of chloroperoxidase compound I is reported which has a peak at 689 nm as its most prominent feature.     

Nucleotide sequence of cytoplasmic initiator tRNA from Tetrahymena thermophila.

The total primary structure of cytoplasmic initiator tRNA from Tetrahymena thermophila mating type IV, was determined by post labeling techniques. The sequence is pa-G-C-A-G-G-G-U-m1G-G-C-G-A-A-A-D-Gm-G-A-A-U-C-G-C-G-U-Psi-G-G-G-C-U-C-A-U-t6A -A-C-Psi-C-A-A-A-A-m7G-U-m5C-A-G-A-G-G-A-Psi-C-G-m1A-A-A-C-C-U-C-U-C-U-C-U-G-C- U-A-C-C-AOH. The nucleotide residue in the position next to the 5'-end of the anticodon of this tRNA (residue No. 33) is uridine instead of cytidine, which has been found in cytoplasmic initiator tRNAs from multicellular eukaryotic organisms. The sequence of three consecutive G-C base pairs in the anticodon stem common to all other cytoplasmic initiator tRNAs is disrupted in this tRNA; namely, the cytidine at residue 40 in this region is replaced by pseudouridine in Tetrahymena initiator tRNA.     

Effect of charge density of cationic polyelectrolytes on complex formation with DNA

The interaction between DNA and ionen polymers, -[N⁺(CH₃)₂(CH₂)_mN⁺(CH₃)₂(CH₂)_n], with m-n of 3–3, 6–6, and 6–10 were examined in order to know how the binding behavior of cationic polymers with DNA depends on the charge density of polycation. The ionen polymer has no bulky side chain and the binding forces with DNA would be attributed mainly to electrostatic interaction. When 3–3 ionen polymers were added to DNA solution, precipitable complexes with the ratio of cationic residue to DNA phosphate (+/?) of 1/1 and the free DNA molecules were segregated, while 6–6 and 6–10 ionen polymers formed soluble complexes with DNA molecules up to (+/?) = 0.5. This suggests that 3–3 ionen polymers bind cooperatively with DNA while 6–6 and 6–10 ionen polymers bind noncooperatively. The cooperative binding of 3–3 ionen polymer and the noncooperative binding of 6–6 ionen polymer were also supported by the thermal melting and recooling profiles from the midpoint between first and second meltings. It was concluded that the charge density of DNA phosphate is a critical value determining whether the ionen polymers bind to DNA by a cooperative or by a noncooperative binding, since the distance between successive cationic charges of 3–3 ionen polymer is shorter than that between successive phosphate charges on DNA double helix and those of 6–6 and 6–10 ionen polymers are longer.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.