Primary mesenchyme cell-ring pattern formation in 2D-embryos of the sea urchin.

Primary mesenchyme cell (PMC) migration during PMC-ring pattern formation was analyzed using computer-assisted time-lapse video microscopy in spread embryos (2D-embryo) of the sea urchin, Mespilia globulus, and a computer simulation. The PMC formed a near normal ring pattern in the 2D-embryos, which were shown to be an excellent model for the examination of cell behavior in vivo by time-lapse computer analysis. The average migration distance of the ventro-lateral PMC aggregate-forming cells (AFC) and that of the dorso-ventral PMC cable-forming cells (CFC) showed no significant difference. All PMC took a rather straightforward migration path to their destinations with little lag time after ingression. This in vivo cell behavior fitted well to a computer simulation with a non-diffusable chemotaxis factor in the cyber-cell migration field. This simulation suggests that PMC recognize their destination from a very early moment of cell migration from the vegetal plate, and implicates that a chemoattractive region is necessary for making the PMC migration pattern. The left- and right-lateral AFC and dorso and ventral CFC were each derived from an unequally divided one-quarter segment of the vegetal plate. This suggests that AFC and CFC have a distinctive ancestor in the vegetal plate, and the PMC are a heterogeneous population at least in terms of their destination in the PMC-ring pattern.

Heterologous expression of clostridial hydrogenase in the Cyanobacterium synechococcus PCC7942.

The Clostridium pasteurianum hydrogenase I has been expressed in the cyanobacterium Synechococcus PCC7942. The Shine-Dalgarno sequence of the structural gene encoding hydrogenase I from C. pasteurianum was changed to that of the cat (chloramphenicol acetyltransferase) gene. The hydrogenase gene was cloned downstream of a strong promoter, isolated from Synechococcus PCC7942, with the cat gene as a reporter gene. Expression of clostridial hydrogenase was confirmed by Western and Northern blot analyses in Synechococcus and Escherichia coli, whereas in vivo/in vitro measurements and activity staining of soluble proteins separated on non-denaturing polyacrylamide gels revealed functional expression of hydrogenase only in cyanobacterial cells. The changed Shine-Dalgarno sequence appeared to be essential for the functional expression of clostridial hydrogenase in Synechococcus, but had no influence on the expression and activity of clostridial hydrogenase expressed in E. coli.

Association of the sea urchin EGF-related peptide, EGIP-D, with fasciclin I-related ECM proteins from the sea urchin Anthocidaris crassispina.

Exogastrula-inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP-D (EGIP-D-binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP-alpha and EBP-beta) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell-binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC-32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP-D as does native EBP, suggesting the interaction between EGF-related proteins and fasciclin I-related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer.

Preventive behaviors against HIV transmission adopted by Japanese commercial sex workers (CSWs).

To assess self-protective behaviors of commercial sex workers (CSWs) against HIV transmission, a consecutive study was conducted at an obstetric-gynecology clinic in the largest "soapland" area in Tokyo. Among 208 CSWs interviewed, only half (107) regularly requested (RR) the clients to use condoms. Sixty-two percent of RR had learned from others ("learners") oral application of condoms while only 39% of NRR (non-regular requesters) did ("non-learners") (p = 0.001). Seventy-six percent of the learners were only "rarely" or "never" detected by the clients regarding condom application compared with only 23% of the non-learners (p< 0.001). While 90% of additionally recruited 40 NRR reported that they would request condom use by a "suspicious" client, none had experienced violence even when the clients found the secretive application. The oral application of condoms appears to be a transferable and potentially effective preventive behavior.

Axonal growth-related cell surface molecule, neurin-1, involved in neuron-glia interaction.

We purified and characterized a novel axonal growth-related molecule, neurin-1, which is anchored to the surface membrane via a phosphatidylinositol (PI) linkage. This molecule was detected by a combination of phosphatidylinositol-specific phospholipase C (PI-PLC) treatment from detergent-soluble mouse brain membranes and subsequent Western blot analysis with monoclonal antibody (MAb 2A). Neurin-1 is immunologically distinct from other known axonal growth associated surface glycoproteins. In immunoblots of embryonic mouse brain membrane, the MAb 2A recognized a single band at approximately 68 kDa, and showed that neurin-1 is mainly associated with fiber-containing regions of developing embryonic mouse brain. Expression is immunohistochemically similar to that of cell adhesion molecule L1, but in comparison, neurin-1 appears somewhat later. Late in embryonic development, neurin-1 appeared to be more stage- and region-specific. Its precise localization at the neural cell surface membranes was confirmed by immuno-electron microscopy using labeled and cultured live nerve cells. Neurin-1 was found only on the surface of the axon and growth cone. Neurin-1, otherwise termed PI anchor protein, corresponds closely in function to the other PI-anchored cell adhesion molecules. Anti-neurin-1 antibody (MAb 2A), however, perturbs the axonal growth and neural cell migration from the astrocyte feeder layer cultures. These results suggest that neurin-1 is one of the important cell surface molecules mediated in the neuron and glial cell interaction.

Mobility and molecular orientation of vitamin E in liposomal membranes as determined by 19F NMR and fluorescence polarization techniques.

To elucidate the molecular orientation and mobility of alpha-tocopherol (vitamin E) in membranes, 19F nuclear magnetic resonance (NMR) was applied. In bilayer phosphatidylcholine (PC) liposomes, 19F NMR spin-spin relaxation times (T2) of 19F-labeled methyl groups on either the chromanol moiety or the isoprenoid side chain of alpha-tocopherol were significantly decreased compared to those for alpha-tocopherol in solution (CDCl3), suggesting that the vitamin E molecule exists in the lipid core of membranes and that its motional freedom is significantly restricted. The difference in T2 values between the solution state and PC liposomes was greatest for the 5a-CF3 on the chromanol moiety compared with other methyl groups on the isoprenoid side chain. Hence, it is thought that the chromanol moiety of alpha-tocopherol may fit tightly near the surface of the membrane because of its hydrophilicity. When Pr3+ was added to the liposomal suspension, the intensity of the NMR signal for 5a-CF3 changed faster than those of the other methyl groups. When the surface of the liposomal membranes was positively charged using stearyl amine, the mobility of the 5a-methyl group, expressed as the correlation time (tau c), was quite similar to that in negatively charged membranes. These results indicate that the chromanol moiety of alpha-tocopherol may orient near the membrane surface, and that the hydroxyl group may be hydrogen bonded to the ester carbonyl of PC. For further confirmation of this conclusion, time-dependent changes in the fluidity of liposomes caused by the addition of Pr3+ were measured at the surface and inner region of liposomal membranes using a fluorescence polarization technique. The fluidity increased in the surface region and decreased in the inner region of liposomes containing alpha-tocopherol. These results show that when praseodymium ions may pass through the liposomal membrane, the hydrogen bonds between alpha-tocopherol and the ester carbonyl of PC may be broken, leading to the formation of an alpha-tocopherol-Pr3+ complex, thus causing the fluidity to increase. Based on the findings obtained, mobility and molecular orientation of vitamin E in membranes are proposed.

A birefringence, electron microscopy, and histochemical survey of chromosomal structure in the sperm nuclei of an echinothurid sea urchin, Araeosoma owstoni.

The mature sperm head of Araeosoma owstoni, an echinothurid sea urchin, showed positive birefringence reflecting that the overall orientation of DNA molecules was semiperpendicular toward the nuclear axis of the sperm head. Transmission electron microscopical observation of sperm in this species revealed a highly electron-dense cylindrical coil with an empty central core extending along the major axis of the sperm head. This coil had seven to eight turns along its entire length of 3.5 micron. The maximum width was 0.35 micron near the distal end of the nucleus, and the minimum width was 0.17 micron near the apical end. Lamellar substructures were also present in the sperm nucleus, appearing at the periphery of the electron-dense cylinder in a radial manner. Staining with Feulgen's reaction and acid-orcein indicated that the coil was probably composed of sperm chromosomes.

Metabolism of ipriflavone (TC-80) in rats.

Metabolic studies of ipriflavone (TC-80) in rats by gas-liquid chromatography-mass spectrometry led to the characterization of the following metabolites: the parent compound, 7-hydroxy-3-phenyl-4H-1-benzopyran-4-one, 7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, 3-(4-hydroxyphenyl)-7-isopropoxy-4H-1-benzopyran-4-one, 2-(3-phenyl-4-oxo-4H-1-benzopyran-7-yl)oxypropionic acid, 2-[3-(4-hydroxyphenyl)-4-oxo-4H-1-benzopyran-7-yl]oxypropionic acid and 2-[3-(3-hydroxyphenyl)-4-oxo-4H-1-benzopyran-7-yl]oxypropionic acid. From the metabolites identified, TC-80 was shown to be metabolized primarily by oxidation. In vitro study using tissue slices of rats indicated that the above metabolic changes occurred exclusively in the liver. It was also demonstrated that the compound did not undergo metabolic conversion by gut flora of rats.

Disposition of ipriflavone (TC-80) in rats and dogs.

Oral 14C-ipriflavone was absorbed by rats to give a maximum plasma 14C level at 1.5 h and a half-life of 5.8 h. In dogs, after po dosing, the plasma 14C peaked at 0.5 h, followed by gradual decline. The plasma of both animals contained mostly metabolites, with small amounts of unchanged ipriflavone. In rats, 14C was distributed widely in tissues, with relatively high concns. in the liver, kidney and gut. Distribution in rat thigh bone of unmetabolized ipriflavone was also demonstrated. 14C-Ipriflavone was eliminated mostly as metabolites within 48 and 72 h, respectively, in rats and dogs. Rats excreted more 14C in urine than in feces, whereas the reverse was noted in dogs. Biliary excretion and reabsorption of 14C were also obvious in both animals.

Studies on caerulein (FI6934). Absorption, distribution, metabolism and excretion of caerulein.

35S-Caerulein (35S-FI6934) was administered intramuscularly into rats (280 mug/kg), rabbits (380 mug/kg) and mice (3.3 mg/kg). Blood level of radioactivity in rats and rabbits reached the maximum at 5 and 15 min after administration, respectively, and then decreased rapidly. In both rats and rabbits, the radioactivities were excreted mainly into the urine. The physiological activity of FI6934 was detected in the blood of rats and rabbits collected within 15 and 30 min after injection respectively, and in the bile of rats collected within the first 2 hr. In rats, the radioactivities were densely distributed in kidney, liver, pancreas, and intestine. Four metabolites of 35S-FI6934 were isolated by paper chromatography from rat urine (i.m., 500 mug/kg). The main metabolites, F-I and F-II, were negative to nynhydrin and to the Ehrlich reagent and carried no physiological activity. 35S-Chym-I, which was prepared by digesting 35S-FI6934 with chymotrypsin, was injected to rats (i.m. 16 or 8 mg/kg/. The metabolite, C-I, isolated from the urine was considered to have a very similar structure to F-I from the results of paper chromatography and paper electrophoresis. By amino acid analysis, the structure of C-I was estimated to be as follows: Pyr-Gln (or Glu)-Asp-Tyr-Thr-Gly. (see article).