Physiological Role of Leghaemoglobin Heterogeneity in Pea Root Nodule Development

Disk-gel-electrophoresis of the leghaemoglobin isolated frompea root nodules revealed two major (Lb I and Lb IV) and threeminor components. The ratio of the two major components (LbI/Lb IV) decreased with increasing age. This ratio was higherin the distal than in proximal region when nodules were cutinto distal and proximal sections. In young nodules, the incorporationof radioactive leucine into Lb I was much higher than into LbIV. In older nodules, the radioactivities were much higher inLb IV than in Lb I. These data suggest that the change in thisratio is due mainly to differences in the rates of biosynthesis. Two major components (Lb I and Lb IV) which were isolated separatelyhad different O₂-binding affinities. The O₂-binding affinityof the component synthesized mainly in older nodules (Lb IV)was higher than that of the component synthesized mainly inyoung nodules (Lb I). These results indicate that changes inthe relative contents of leghaemoglobin during nodule developmentcontribute to more effective nitrogen fixation which is mediatedby changes in the capacities of oxygen transport. (Received July 7, 1981; Accepted November 2, 1981)     

The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif

The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.     

Adductor muscles of the thigh of crab-eating monkeys (Macaca fascicularis)

On the basis of 44 hindlimbs of 14 male and 14 female crab-eating monkeys (Macaca fascicularis), the morphology of the adductor muscles of the thigh was described and some functional indices were calculated. The results obtained from this study agreed generally with those of otherMacaca species reported by various authors. For the classification and nomenclature of the adductors, the criteria proposed byUhlmann (1967, 1968) was well adapted to the crab-eating monkey. The adductors comprise the m. gracilis, m. pectineus, m. adductor longus, pars longa and pars brevis of m. adductor brevis, pars lata and pars minima of m. adductor magnus and m. obturatorius externus. In males, the adductors are generally inserted further down the femur, and the insertions of pars brevis of the m. adductor brevis and pars minima of the m. adductor magnus have longer attachments to the femur than in females. The arrangement of each adductor muscle and of each fasciculus of a thigh muscle may invoke a principle of organization.     

Efficient Conversion of L-Tryptophan to Indole-3-Acetic Acid and/or Tryptophol by Some Species of Rhizoctonia

In a study of various phytopathogenic fungi, we found that fungithat belong to the genus Rhizoctonia produce IAA efficientlyfrom tryptophan. R. solani Kühn MAFF-305219, in particular,produced large amounts of tryptophol (Tol), which was assumedto be a specific by-product of the indole-3-pyruvate (IPy) pathway,in addition to IAA. Therefore, this fungus seemed suitable foranalysis of the function and the regulation of the biosynthesisof auxin by a fungal pathogen. Under normal aerobic conditions,the ratio of IAA to Tol synthesized by this strain was higherthan that under less aerobic conditions. In metabolic studieswith various indole derivatives, R. solani converted L-tryptophanand indole-3-acetaldehyde to IAA and Tol, but other indole derivativeswere scarcely metabolized. These results suggest that both IAAand Tol are synthesized from tryptophan through the IPy pathwayin Rhizoctonia. (Received May 27, 1996; Accepted July 8, 1996)     

Analysis of the cellular heterogeneity in the basal layer of mouse ear epidermis: an approach from partial decomposition in vitro and retroviral cell marking in vivo

In the thin epidermis, the existence of epidermal proliferation units was hypothesized. Each unit is supposed to be partitioned into each column of polygonal-shaped cornified plates, estimated to contain a central stem cell in its basal layer. We attempted to verify this hypothesis in vitro by analyzing the partially decomposed fragment of mouse ear epidermis and in vivo using retroviral cell marking. Partially decomposed fragments of the mouse ear epidermis, mostly composed of cytokeratin 14-expressing basal keratinocytes, formed multicellular colonies in vitro. They were composed of heterogeneously shaped cells, morphologically resembling the cells in each single cell-derived colony, including potential stem cells with great proliferative potency in vitro. The estimated frequency of the candidates of stem cells in the fragments was much lower than the prediction from the representative hypothesis. Retroviral cell marking with nuclear localizing LacZ protein in vivo suggested the existence of a large clonal cellular unit for epidermal renewal. From these in vitro and in vivo observations, we propose a new model for the epidermal proliferation unit.     

Effects of PAX3-FKHR on malignant phenotypes in alveolar rhabdomyosarcoma

The malignancy of alveolar rhabdomyosarcoma (ARMS) has been linked to expression of the PAX3-FKHR chimeric gene. To understand the effect of this gene, we used RNAi to knock down its expression (without affecting the expressions of either PAX3 or FKHR) in human ARMS cell lines. Down-regulating PAX3-FKHR caused (a) tumor cells to accumulate in the G1 phase, inhibiting the rate of cellular proliferation, (b) a reduction in the levels of the MET, reducing cell motility stimulated by HGF, and (c) induction of the myogenic differentiation gene, myogenin, and muscle differentiation (morphologic change and the expression of muscle specific proteins, desmin, and myosin heavy chain). These results suggest that PAX3-FKHR in ARMS cells promotes malignant phenotypes such as proliferation, motility, and to suppress differentiation.     

Reduction of Aluminum Toxicity by Addition of a Conditioned Medium from Aluminum-Tolerant Cells of Carrot

The toxic effect of aluminum (Al) on the growth of Carrot cells(SO-l) decreased to a greater degree with addition of a mediumconditioned by Al-tolerant carrot cells (TA-l) than with a mediumconditioned by SO-l cells. The toxic effect of Al was reducedgreatly by adding an acidic fraction of the conditioned media,but little or not at all by a neutral or basic fraction. Offour organic acids detected in the acidic fraction, the majorone was citric acid which was present in a much greater amountin the conditioned medium of TA-l cells than in that of SO-lcells. The toxic effect of Al was reduced by adding citric or malicacid instead of the conditioned medium, but not by succinicor fumaric acid. Chelating abilities of the organic acids wereevaluated by shifts in their titration curves, and were foundto be closely correlated with the detoxification effects. Thus,the Al tolerance of TA-l cells may in fact be due to the chelatingeffect of citric acid which is abundantly released into themedium by the Al-tolerant carrot cells. (Received July 9, 1984; Accepted November 22, 1984)     

Synchronous progression of calcium transient-dependent beating and sarcomere destruction in apoptotic adult cardiomyocytes

During early apoptosis, adult cardiomyocytes show unusual beating, suggesting possible participation of abnormal Ca(2+) transients in initiation of apoptotic processes in this cell type. Simultaneously with the beating, these cells show dynamic structural alteration resulting from cytoskeletal disintegration that is quite rapid. Because of the specialized structure and extensive cytoskeleton of cardiomyocytes, we hypothesized that its degradation in so short a time would require a particularly efficient mechanism. To better understand this mechanism, we used serial video microscopy to observe beta-adrenergic stimulation-induced apoptosis in isolated adult rat cardiomyocytes while simultaneously recording intracellular Ca(2+) concentration and cell length. Trains of Ca(2+) transients and corresponding rhythmic contractions and relaxations (beating) were observed in apoptotic cells. Frequencies of Ca(2+) transients and beating gradually increased with time and were accompanied by cellular shrinkage. As the cells shrank, amplitudes of Ca(2+) transients declined and diastolic intracellular Ca(2+) concentration increased until the transients were lost. Beating and progression of apoptosis were significantly inhibited by antagonists against the L-type Ca(2+) channel (nifedipine), ryanodine receptor (ryanodine), inositol 1,4,5-trisphosphate receptor (heparin), sarco(endo)plasmic Ca(2+)-ATPase (thapsigargin), and Na(+)/Ca(2+) exchanger (KB-R7943). Electron-microscopic examination of beating cardiomyocytes revealed progressive breakdown of Z disks. Immunohistochemical analysis and Western blot confirmed that disappearance of Z disk constituent proteins (alpha-actinin, desmin, and tropomyosin) preceded degradation of other cytoskeletal proteins. It thus appears that, in adult cardiomyocyte apoptosis, Ca(2+) transients mediate apoptotic beating and efficient sarcomere destruction initiated by Z disk breakdown.