Monoclonal antibody to human plasma gelsolin.

Human plasma gelsolin was purified by column chromatography. The method yielded a protein of high purity and activity. Using this protein, we produced monoclonal antibody (Mab H6B11) against human plasma gelsolin by somatic cell fusion. This monoclonal antibody reacted in a dose-dependent manner with gelsolin derived from human plasma and platelets and neutralized depolymerizing activity to F-actin. It differed from the commercially available substance (Mab G4896; Sigma) in that the time required for the reaction between the antigen and antibody in the enzyme-linked immunosorbent assay could be shortened by one-third. The antibody was judged to be useful in assays for elucidating the physiological role of plasma gelsolin.     

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.     

Synthesis of sperm-specific basic nuclear proteins (SPs) in cultured spermatids from Xenopus laevis

The accumulation and synthesis of sperm-specific basic nuclear proteins (SPs) in Xenopus spermatids in vitro were studied by acid-urea-Triton polyacrylamide gel electrophoresis and fluorography. In synchronous cultures of round spermatids, the amount of SP2 and SP3-5 accumulated almost linearly with time, while that of SP1 remained almost constant. Fluorography showed that round spermatids incorporated [14C]arginine mostly into SP1 and SP3-5, very little into SP2, and none into histones. When [14C]arginine was incorporated into cells for 24 h on Days 0, 3, and 6, followed by immediate extraction of basic nuclear proteins, the SP1 band was detected faintly on Day 0 and the intensity increased to the maximum level by Day 3 and remained constant on Day 6; the SP3-5 bands were first detected on Day 3 and their intensity increased by Day 6. Thus, SP1 and SP3-5 were synthesized differentially during the culture period. When [14C]arginine or [14C]lysine was incorporated into round spermatids on Days 0, 3, and 6 for 15 h and chased for 3-12 days, the intensity of the SP2 band increased significantly, while the intensity of the SP1 band decreased concomitantly. This result indicates that SP2 was processed from a precursor protein which is probably SP1.     

Mechanisms to Evade the Phagocyte Respiratory Burst Arose by Convergent Evolution in Typhoidal Salmonella Serovars

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Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons

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Accumulation of Ascorbic Acid in the Cotyledons of Morning Glory (Pharbitis nil) Seedlings during the Induction of Flowering by Low-Temperature Treatment and the Effect of Prior Exposure to High-Intensity Light

Seedlings of Pharbitis nil strain ‘Violet’ werecultured at a low temperature, which induces their floweringeven in continuous light, with or without prior exposure tohigh-intensity light, which enhances the flower-inducing effectof the exposure to low temperature. Analysis by HPLC of extractsof cotyledons showed that the level of an unstable compoundincreased during these treatments, in addition to the increasein levels of phenylpropanoids reported previously. The compoundwas identified as ascorbic acid from the spectroscopic data.The change in the concentration of ascorbic acid at low temperaturewas correlated with the increase in the induction of floweringand the increase in levels of the phenylpropanoids. The rapidincrease in level of ascorbic acid after exposure to high-intensitylight reflected the promotive effect of high-intensity lighton the induction of flowering at low temperature. However, levelsof ascorbic acid also increased in seedlings of P. nil strain‘Kidachi’ that were cultured in high-intensity light,a treatment that does not induce flowering in this strain. Thus,ascorbic acid cannot be associated with the induction of floweringby high-intensity light alone. Ascorbic acid increased the rateof formation of caffeic acid from p-coumaric acid in vitro,a result that suggests that ascorbic acid might be involvedin the increases in levels of phenylpropanoids in the seedlings. (Received April 17, 1995; Accepted August 1, 1995)     

Experimental Chlamydia psittaci Infection of Japanese Quail

Japanese quail were used for the infection model of avian chlamydiosis. One-day-old Japanese quail were highly susceptible to lethal infection by a Chlamydia psittaci strain of budgerigar origin upon inoculation via the air sac route with 10^4.1 FFU of the organism, showing an acute and lethal course with chlamydial propagation. In contrast, 7-day-old quail developed resistance to the infection as shown by the lack of lethal effect with the same dose. The resistance of 7-day-old birds was abolished by immunosuppressive treatment with cyclophosphamide. Upon inoculation with a sublethal dose of 10^2.1 FFU, latent infection was established in 1-day-old birds with a minimum number of the organism. The latent infection in the birds was converted to the lethal form by treatment with cyclophosphamide along with chlamydial propagation and suppression of antibody production.     

Isoproterenol directs hair follicle-associated pluripotent (HAP) stem cells to differentiate in vitro to cardiac muscle cells which can be induced to form beating heart-muscle tissue sheets

Nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells are located in the bulge area of the follicle. Previous studies have shown that HAP stem cells can differentiate to neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. HAP stem cells effected nerve and spinal cord regeneration in mouse models. Recently, we demonstrated that HAP stem cells differentiated to beating cardiac muscle cells. The differentiation potential to cardiac muscle cells was greatest in the upper part of the follicle. The beat rate of the cardiac muscle cells was stimulated by isoproterenol. In the present study, we observed that isoproterenol directs HAP stem cells to differentiate to cardiac muscle cells in large numbers in culture compared to HAP stem cells not supplemented with isoproterenol. The addition of activin A, bone morphogenetic protein 4, and basic fibroblast growth factor, along with isoproternal, induced the cardiac muscle cells to form tissue sheets of beating heart muscle cells. These results demonstrate that HAP stem cells have great potential to form beating cardiac muscle cells in tissue sheets.     

Early-age-dependent selective decrease of differentiation potential of hair-follicle-associated pluripotent (HAP) stem cells to beating cardiac-muscle cells

We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.     

Involvement of Norepinephrine in the Production of a Flower-Inducing Substance in the Water Extract of Lemna

A flower-inducing substance (FIS) is produced by incubationof the pellet of centrifuged ho-mogenates of Lemna paucicostata441 (P441) with commercial enzyme preparations such as catalase,cellulase, lipase and proteinase K. The active component inthese preparations was identified as tyrosine. The tyrosinemetabolites, dopa, dopamine, norepinephrine (NE) and epinephrine,were also effective for the production of FIS, and NE the mosteffective. Addition of only 1 µg NE to the pellet obtainedfrom 100 mg fresh weight of P441 produced FIS without incubation.NE added to the pellet heated after resuspension also producedFIS but that added to the pellet resuspended in boiling waterimmediately after separation did not. A heat-stable substance(s)produced by a heat-unstable reaction in the pellet may reactwith NE to produce FIS or interact with NE to induce flowering.About 400 ng per g fresh weight of NE was detected in the waterextract of P441 plants, but only about 0.5 ng in the hot-acidicwater extract of the plants, which suggests that most of theNE was produced after homogenization. (Received October 17, 1990; Accepted December 28, 1990)