Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Identification of phytochrome B amino acid residues mutated in three new phyB mutants of Arabidopsis thaliana

The growth and development of plants is regulated by light viathe action of photoreceptors which are responsive to the red/far-red,blue and UV regions of the spectrum. Phytochrome B (the apoproteinof which is encoded by the PHYB gene) is one of the red/far-redabsorbing photoreceptors active in this process. In this paper,the isolation and characterization of three new EMS-inducedmutations of Arabidopsis which confer phytochrome B deficiencyare described. Complementation analysis showed that these mutations(phyB-101, phyB-102 and phyB-104) were allelic with PHYB. DNAsequence analysis showed that all three mutants contain nucleotidesubstitutions in the PHYB-101 gene sequence. phyB-101 carriesa nucleotide substitution within the second exon of the PHYBgene. This G-to-A substitution is a missense mutation that convertsa glutamate residue at position 812 of the phytochrome B apoproteinto a lysine residue. phyB-102, another missense mutant, carriesa C-to-T substitution which converts a serine residue at position349 of the phytochrome B apoprotein to a phenylalanine residue.phyB-104 carries a premature stop codon as a result of a G-to-Amutation 1190 bp down-stream of the ATG start codon of the PHYBsequence. The missense mutations in phyB-101 and phyB-102 causesignificant alterations in the predicted second ary structureof their respective mutant polypeptides, and identify aminoacid residues playing crucial roles in phytochrome B function,assembly or stability. Key words: Arabidopsis thaliana, phytochromet, phyB mutants, missense mutations     

Relationship between tumor necrosis factor alpha and feline immunodeficiency virus expressions.

The presence of feline immunodeficiency virus (FIV) proviral DNA, expression of FIV p26 core protein, and production of tumor necrosis factor alpha (TNF-alpha) were assessed in sequential biopsies of spleen and lymph node sections, of mononuclear cells of the peripheral blood, and of the serum of specific-pathogen-free cats during the acute phase of FIV infection. A temporal relationship between TNF-alpha production and FIV p26 expression was noted. Two months following FIV infection, and preceding the detection of FIV viremia, levels of TNF-alpha in serum increased significantly (P = 0.04), and they remained elevated during FIV viremia in the third month postinfection. Immunoprecipitates representing expression of TNF-alpha and of FIV p26 were localized in common foci of lymph nodes of FIV-infected cats during this period of active viremia. With the advent of anti-FIV antibodies, circulating levels of TNF-alpha and p26 antigen and expression of TNF-alpha and p26 in the lymph nodes decreased during the fifth month postinfection, and p26 production became undetectable. With clearance of viremia, burden of proviral DNA in peripheral blood mononuclear cells became reduced (P = 0.041), with provirus remaining integrated principally within lymph nodes (P = 0.046). During aviremia, p26 expression was undetectable in any tissue but remained inducible in vitro. During acute FIV infection, TNF-alpha production and p26 expression are intimately linked.     

Changes in leaf expansion and epidermal screening effectiveness in Liquidambar styraciflua and Pinus taeda in response to UV-B radiation

Absorption or screening of ultraviolet-B (UV-B) radiation by the epidermis may be an important protective method by which plants avoid damage upon exposure to potentially harmful UV-B radiation. In the present study we examined the relationships among epidermal screening effectiveness, concentration of UV-absorbing compounds, epidermal anatomy and growth responses in seedlings of loblolly pine (Pinus taeda L.) and sweetgum (Liquidambar styraciflua L.). Seedlings of each species were grown in a greenhouse at the University of Maryland under either no UV-B radiation or daily supplemental UV-B radiation levels of 4, 8 or 11 kJ m^?2 of biologically effective UV-B (UV-B_BE) radiation. Loblolly pine seedlings were subsequently grown in the field under either ambient or supplemental levels of UV-B radiation. At the conclusion of the growing season, measurements of epidermal UV-B screening effectiveness were made with a fiber-optic microprobe. In loblolly pine, less than 0.5% of incident UV-B radiation was transmitted through the epidermis of fascicle needles and about 1% was transmitted in primary needles. In contrast, epidermal transmittance in sweetgum ranged from about 20% in leaves not preconditioned to UV-B exposure, to about 10% in leaves grown under UV-B radiation. The concentration of UV-absorbing compounds was unaffected by UV-B exposure, but generally increased with leaf age. Increases in epidermal thickness were observed in response to UV-B treatment in loblolly pine, and this accounted for over half of the variability in UV-B screening effectiveness. In spite of the low levels of UV-B penetration into the mesophyll, delays in leaf development (both species) and final needle size (loblolly pine) were observed. Seedling biomass was reduced by supplemental UV-B radiation in loblolly pine. We hypothesize that the UV-induced growth reductions were manifested by changes in either epidermal anatomy or epidermal secondary chemistry that might negatively impact cell elongation.     

Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci.

We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.     

Cobalt(II) Oxidation by the Marine Manganese(II)-Oxidizing Bacillus sp. Strain SG-1

The geochemical cycling of cobalt (Co) has often been considered to be controlled by the scavenging and oxidation of Co(II) on the surface of manganese [Mn(III,IV)] oxides or manganates. Because Mn(II) oxidation in the environment is often catalyzed by bacteria, we have investigated the ability of Mn(II)-oxidizing bacteria to bind and oxidize Co(II) in the absence of Mn(II) to determine whether some Mn(II)-oxidizing bacteria also oxidize Co(II) independently of Mn oxidation. We used the marine Bacillus sp. strain SG-1, which produces mature spores that oxidize Mn(II), apparently due to a protein in their spore coats (R.A. Rosson and K. H. Nealson, J. Bacteriol. 151:1027-1034, 1982; J. P. M. de Vrind et al., Appl. Environ. Microbiol. 52:1096-1100, 1986). A method to measure Co(II) oxidation using radioactive ⁵⁷Co as a tracer and treatments with nonradioactive (cold) Co(II) and ascorbate to discriminate bound Co from oxidized Co was developed. SG-1 spores were found to oxidize Co(II) over a wide range of pH, temperature, and Co(II) concentration. Leucoberbelin blue, a reagent that reacts with Mn(III,IV) oxides forming a blue color, was found to also react with Co(III) oxides and was used to verify the presence of oxidized Co in the absence of added Mn(II). Co(II) oxidation occurred optimally around pH 8 and between 55 and 65°C. SG-1 spores oxidized Co(II) at all Co(II) concentrations tested from the trace levels found in seawater to 100 mM. Co(II) oxidation was found to follow Michaelis-Menten kinetics. An Eadie-Hofstee plot of the data suggests that SG-1 spores have two oxidation systems, a high-affinity-low-rate system (K_m, 3.3 × 10^-8 M; V_max, 1.7 × 10^-15 M · spore^-1 · h^-1) and a low-affinity-high-rate system (K_m, 5.2 × 10^-6 M; V_max, 8.9 × 10^-15 M · spore^-1 · h^-1). SG-1 spores did not oxidize Co(II) in the absence of oxygen, also indicating that oxidation was not due to abiological Co(II) oxidation on the surface of preformed Mn(III,IV) oxides. These results suggest that some microorganisms may directly oxidize Co(II) and such biological activities may exert some control on the behavior of Co in nature. SG-1 spores may also have useful applications in metal removal, recovery, and immobilization processes.     

Subunits of forming pollen exine and Ubisch bodies as seen in freeze substitutedLedebouria socialis Roth (Hyacinthaceae)

Summary High-pressure freezing/freeze substitution/TEM was employed to investigate anthers of the monocotyledonous angiospermLedebouria socialis Roth (Hyacinthaceae) during early tetrad stage. The initials of the outer sporopollenous pollen wall stratum (=sexine) and of the homologous tapetal products (=Ubisch bodies) are composed of highly regular subunits: clustered globules with a constant diameter of approximately 28 nm. The clusters develop within diffuse accumulations of electron-dense material. This process, interpreted as sporopollenin polymerization, does not necessarily depend on the presence of membrane-bound enzymes. Immunogold labeling with JIM 5 and JIM 7 antibodies revealed that the primexine as well as the dissolving tapetal cell walls, the sites of sexine and Ubisch body formation, respectively, contain un-esterified and methyl-esterified pectins.Abbreviations E-PTA ethanolic phosphotungstic acid - PA periodic acid - UA/Pb uranyl acetate/lead     

The world-wide web and plant molecular biology

The Internet has been functional since 1967 and has been operating without interruption for over 20 years. Although local service can be lost, the network will retain its integrity and recover from almost any imaginable combination of faults, whether natural or intentional. This robustness, the global scope, the availability of quality public domain software, and the inherent democracy of the Internet, have combined to eclipse similar efforts. Many scientists have found the Internet immediately useful for communication in the form of electronic mail. Shared resources, an intrinsic benefit of networks, are now becoming apparent in the form of the World-Wide Web (WWW).     

Characterization of seminal plasma proteins and sperm proteins in ejaculates from normospermic bulls and bulls with thermally-induced testicular degeneration

Semen was collected from 5 mature beef bulls by electroejaculation before, during, and after 20 days of scrotal insulation. Thermally-induced testicular degeneration was irreversible in 3 of the bulls. Analysis of sperm and seminal plasma polypeptides revealed that 15 to 30 sperm polypeptides and 25 to 30 seminal plasma polypeptides were indistinguishable between bulls prior to the insulation treatment. Changes in the sperm polypeptides pattern appeared as early as 2 days after the insulation treatment and persisted for at least 11 months in 2 of the bulls. In the spermatozoa, there was a detectable loss of 31, 34, 49 and 58 kDa polypeptides and an appearance of 6 to 8 new major polypeptides, ranging from 32 to 83 kDa. The 83 kDa polypeptide was most prominent in the 2 bulls that regained normal sperm motility and morphology following the insulation period. The post-insulation appearance of a seminal plasma polypeptide (circa 60 kDa) was also identified in these 2 bulls. Seminal plasma polypeptides remained qualitatively unaltered by the insulation treatment in the 3 bulls with irreversible testicular degeneration.