Improvement of Sperm Motility of Sex-Reversed Male Rainbow Trout, Oncorhynchus mykiss, by Incubation in High-pH Artificial Seminal Plasma

Changes in the motility time of spermatozoa collected from the testes and the sperm duct of normal and sex-reversed male (XX) rainbow trout in physiological balanced salt solution were examined after incubation in artificial seminal plasmas of various pHs. Although untreated spermatozoa from the sperm duct retained motility for 60–90 s in the balanced salt solution, the spermatozoa collected from the testes were immotile. During the incubation in artificial seminal plasma of pH 7.0, the spermatozoa from the sperm duct hardly moved, similar to the testicular spermatozoa in the balanced salt solution. By suspending and incubating the testicular spermatozoa in artificial seminal plasma of pH 9.9 for 2 h at 4°C, the percentage of motile spermatozoa increased from 0–5% to 80%. The spermatozoa remained motile for at least 2 min after long-term incubation (12 h). When the full-sib eggs were inseminated with untreated testicular spermatozoa or testicular sperm treated for 2 h at high pH, the percentage survival increased from 5.5% to 53.8% at the eyed stage due to the high-pH treatment. The incubation of the spermatozoa in high-pH artificial seminal plasma improved the motility of the spermatozoa from the testes of the sex-reversed male that had lost its sperm duct. By this treatment, it is possible to markedly increase the mass production efficiency of all-female or all-female triploid sterile progenies.     

Development of cell-expressed and virion-incorporated CCR5-targeted vaccine

Our previous study demonstrated that the immunization with a cycloimmunogen derived from extracellular loop-2 (ECL-2) of CCR5 (cDDR5) attenuated acute phase of CCR5-tropic simian-human immunodeficiency virus (SHIV)_SF162P3 replication in vivo. Although the study showed that the antisera raised against cDDR5 reacted with cell-expressed CCR5, we have not yet demonstrated whether the antisera can react with virion-incorporated CCR5. Here, we show that rhesus cDDR5 (rcDDR5)-specific antibodies react with not only cell-expressed but also virion-incorporated simian CCR5s (siCCR5s), but may predominantly exert their inhibitory effects on simian immunodeficiency virus (SIV) infection by the binding of cell-expressed rather than virion-incorporated CCR5s. These results suggest that the virion-incorporated CCR5 may contribute to the reactivation of the anti-rcDDR5 antibody-producing B-cells by SIV particles after rcDDR5 immunization, although the binding of anti-rcDDR5 antibody to virion-incorporated CCR5 results in a partial inhibitory effect on SIV infection.     

Stability and ferrous iron content of the nitrogenase and its components from Azotobacter vinelandii

Summary Nitrogenase of Azotobacter vinelandii is sensitive to oxygen, and sensitivity develops during purification. Such inactivation is well prevented by 0.1% hydroquinone plus 0.01% ascorbate, which are also effective in preventing inactivation of enzyme on storage under H₂. Activity is proportional to ferrous iron content of crude sample of enzyme. On storage at 0°C, 0.3 M KCl inactivates the enzyme, while KCl stabilizes its components. Nitrogenase is not cold labile, while the components are cold labile; Fe, Mo-component is most stable at 22°C and Fe-component at 13.5°C. Nitrogenase substrates, except N₂, stabilize nitrogenase, but not the components.     

Aminooxy‐naphthylpropionic acid and its derivatives are inhibitors of auxin biosynthesis targeting l‐tryptophan aminotransferase: structure–activity relationships

We previously reported l ‐α‐aminooxy‐phenylpropionic acid (AOPP) to be an inhibitor of auxin biosynthesis, but its precise molecular target was not identified. In this study we found that AOPP targets TRYPTOPHAN AMINOTRANSFERASE of ARABIDOPSIS 1 (TAA1). We then synthesized 14 novel compounds derived from AOPP to study the structure–activity relationships of TAA1 inhibitors in vitro. The aminooxy and carboxy groups of the compounds were essential for inhibition of TAA1 in vitro. Docking simulation analysis revealed that the inhibitory activity of the compounds was correlated with their binding energy with TAA1. These active compounds reduced the endogenous indole‐3‐acetic acid (IAA) content upon application to Arabidopsis seedlings. Among the compounds, we selected 2‐(aminooxy)‐3‐(naphthalen‐2‐yl)propanoic acid (KOK1169/AONP) and analyzed its activities in vitro and in vivo. Arabidopsis seedlings treated with KOK1169 showed typical auxin‐deficient phenotypes, which were reversed by exogenous IAA. In vitro and in vivo experiments indicated that KOK1169 is more specific for TAA1 than other enzymes, such as phenylalanine ammonia‐lyase. We further tested 41 novel compounds with aminooxy and carboxy groups to which we added protection groups to increase their calculated hydrophobicity. Most of these compounds decreased the endogenous auxin level to a greater degree than the original compounds, and resulted in a maximum reduction of about 90% in the endogenous IAA level in Arabidopsis seedlings. We conclude that the newly developed compounds constitute a class of inhibitors of TAA1. We designated them ‘pyruvamine’.     

Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.

Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))     

Occurrence of Potential Brassinosteroid Precursor Steroids in Seeds of Wheat and Foxtail Millet

Triticum aestivum L.) and foxtail millet (Setaria italica Beauv.) were found by GC-MS to contain, in addition to bulk sterols, 4-en-3-one steroids including 24-ethylcholesta-4,24(28)Z- dien-3-one (a new steroid), 24-methylcholest-4-en-3-one, 24-ethylcholesta-4,22E-dien-3-one and 24-ethylcholest-4-en-3-one, as well as 5α-steroidal 3-one compounds including 24-methyl-5α-cholestan-3-one, 24-ethyl-5α-cholestan-3-one and 24-ethyl 5α-cholest-22E-en-3-one (in S. italica only). Analysis of free sterol and steryl ester fractions indicated that campestanol and sitostanol were present at high levels in both seeds. These results suggest that the seeds of T. aestivum and S. italica synthesize campestanol from campesterol via 24-methylcholest-4-en-3-one and 24-methyl-5α-cholestan-3-one as has already been demonstrated in Arabidopsis thaliana L., and also produce sitostanol from sitosterol via 24-ethylcholest-4-en-3-one and 24-ethyl-5α-chotestan-3-one. Biosynthetic relationships of campestanol and sitostanol with C₂₈ and C₂₉ brassinosteroids are discussed. Received 4 September 1998/ Accepted in revised form 26 November 1998     

Molecular basis of spectral tuning in the red- and green-sensitive (M/LWS) pigments in vertebrates

Vertebrate vision is mediated by five groups of visual pigments, each absorbing a specific wavelength of light between ultraviolet and red. Despite extensive mutagenesis analyses, the mechanisms by which contemporary pigments absorb variable wavelengths of light are poorly understood. We show that the molecular basis of the spectral tuning of contemporary visual pigments can be illuminated only by mutagenesis analyses using ancestral pigments. Following this new principle, we derive the "five-sites" rule that explains the absorption spectra of red and green (M/LWS) pigments that range from 510 to 560 nm. Our findings demonstrate that the evolutionary method should be used in elucidating the mechanisms of spectral tuning of four other pigment groups and, for that matter, functional differentiations of any other proteins.