Early development and neurogenesis of Temnopleurus reevesii

Sea urchins are model non‐chordate deuterostomes, and studying the nervous system of their embryos can aid in the understanding of the universal mechanisms of neurogenesis. However, despite the long history of sea urchin embryology research, the molecular mechanisms of their neurogenesis have not been well investigated, in part because neurons appear relatively late during embryogenesis. In this study, we used the species Temnopleurus reevesii as a new sea urchin model and investigated the detail of its development and neurogenesis during early embryogenesis. We found that the embryos of T. reevesii were tolerant of high temperatures and could be cultured successfully at 15–30°C during early embryogenesis. At 30°C, the embryos developed rapidly enough that the neurons appeared at just after 24 h. This is faster than the development of other model urchins, such as Hemicentrotus pulcherrimus or Strongylocentrotus purpuratus. In addition, the body of the embryo was highly transparent, allowing the details of the neural network to be easily captured by ordinary epifluorescent and confocal microscopy without any additional treatments. Because of its rapid development and high transparency during embryogenesis, T. reevesii may be a suitable sea urchin model for studying neurogenesis. Moreover, the males and females are easily distinguishable, and the style of early cleavages is intriguingly unusual, suggesting that this sea urchin might be a good candidate for addressing not only neurology but also cell and developmental biology.     

Temperature-gradient DNA probe chromatography of nucleic acids on nonporous supports.

Temperature-gradient DNA probe chromatography of nucleic acids on a nonporous support with a homogeneous particle size of 2.5 microns showed a higher base sequence discriminating power and a larger linear capacity than that on a porous support with a larger and less homogeneous particle size. The resolution on the nonporous support was high enough to separate samples with a single-base mismatch of the less destabilizing groups, including G-G and G-T base mismatches, even when it locates very near the end of the probe.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Analysis of responses to endothelins in isolated porcine blood vessels by using a novel endothelin antagonist, BQ-153.

We examined the effects of a novel ETA-selective endothelin (ET) antagonist, BQ-153, on vascular responses to ET-1 and ET-3 in isolated porcine coronary and pulmonary blood vessels, to clarify the roles of ET receptor subtypes in the regulation of vascular smooth muscle tension. With endothelium-denuded vascular tissues, the concentration-contraction curve (CCC) for ET-1 appeared as a single sigmoidal shape for all types of tissue. The CCC for ET-1 was antagonized by BQ-153 (2 and 10 microM) in all tissues, but part of the contraction was resistant. The CCC for ET-3 usually consisted of two different phases with higher (first phase) and lower (second phase) sensitivities to the peptide. Only the second phase of CCC for ET-3 was completely inhibited by BQ-153 (2 microM) in all tissues, while the first phase was resistant. The BQ-153-resistant contractile phases of ET-1 and ET-3-induced vasoconstriction appeared to have similar sensitivity in all tissues, and the contractile activity varied with each type of tissue. With endothelium-intact materials, the potencies of ET-1 and ET-3 for endothelium-dependent vasorelaxation in pulmonary artery were almost equivalent. BQ-153 (10 microM) did not inhibit ET-induced vasorelaxation. These results indicate that ET-induced vasoconstriction is mediated not only through ETA but also through ETnonA (probably ETB), and that the relative proportions of the ET-receptor subtypes mediating contractions vary in different vascular areas. In addition, results showed that ET-induced endothelium-dependent vasorelaxation is mediated through ETB.     

Oxygen-18 studies on the oxidative deamination mechanism of alicyclic primary amines in rabbit liver microsomes

The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of ¹⁸O₂ or in a medium containing H₂¹⁸O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.5–336}\text{min}$) and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia.     

PPD-specific proliferative response in humans. I. Analysis of PPD-specific proliferative cells from tuberculous pleurisy patients and healthy controls with monoclonal antibodies specific for human T subsets

Peripheral mononuclear cells (PBL) from tuberculin reaction (TR)-negative tuberculous pleurisy patients proliferated poorly with PPD, while the cells of pleural effusion from these patients showed a proliferative response to PPD as well as did the healthy control PBL. Surface antigens of peripheral blood and pleural effusion were examined by using monoclonal antibodies. The Leu 1-positive cell population can be divided into four groups, namely (1) Leu 1+, Leu2a+, Leu 3a+, (2) Leu 1+, Leu 2a+, Leu 3a-, (3) Leu 1+, Leu 2a-, Leu3a+, and (4) Leu 1+, Leu 2a-, Leu 3a- cell populations. Results of analysis of surface antigens of PPD-specific proliferative cells in peripheral blood and pleural effusion from tuberculous pleurisy patients as well as healthy controls indicate that the PPD-specific proliferative response is mediated by Leu 1+, Leu 2a-, Leu 3a+ cells and Leu 1+, Leu 2a-, Leu3a- cells.