Ultrastructural localization of laminin on in vivo embryonic, neonatal, and adult rat cardiac myocytes and in early rat embryos raised in whole-embryo culture.

The temporal and spatial distribution of the basement membrane component laminin was examined in vivo in developing rat hearts at 11.5 and 15 days of embryonic development (ED), and in neonates and adults, by pre-embedding ultrastructural immunocytochemistry. In addition, the patterns observed at 11.5 days ED were compared to the distribution of laminin in embryos maintained in whole-embryo culture. At 11.5 days ED laminin was localized in punctate patches on the surface of the plasma membrane, with large gaps between areas of staining. The development of myocytes and localization of laminin in the whole embryo-cultured embryos was similar to that found in the in vivo embryos. At 15 days ED, laminin localization was limited to distinct patches of developing extracellular matrix material associated with the sarcolemma. Gaps between areas of localization were shorter than in the 11.5-day hearts. In neonates, distribution of laminin localization was more extensive with fewer gaps and was associated with the developing basement membrane. In adult hearts, laminin was localized along the entire length of the basement membrane and was heaviest in areas of morphological specialization, such as Z-bands, where collagen bundles contacted the sarcolemma.     

Convenient synthesis of 18-hydroxylated cortisol and prednisolone.

18,20-Epoxy-11 beta,17 alpha,20 beta,21-tetrahydroxypregn-4-en-3-one was synthesized by the application of hypoiodite reaction to the cortisol acetonide. The intermediary 18-iodo derivative was converted to the 11-oxo steroid by chromic acid prior to silver ion-assisted solvolysis. Removal of the protective group with hydrochloric acid was finally carried out to give the desired 11 beta,17 alpha,18,21-tetrahydroxypregn-4-ene-3,20-dione as the hemiacetal form. 18,20-Epoxy-11 beta-17 alpha,20 beta,21- tetrahydroxypregna-1,4-dien-3-one was also prepared from prednisolone through a similar reaction sequence.     

Assignment of the human granulocyte colony-stimulating factor receptor gene (CSF3R) to chromosome 1 at region p35-p34.3.

The gene for the granulocyte colony-stimulating factor (G-CSF) receptor (CSF3R) was localized on the p35-p34.3 region of human chromosome 1 by in situ hybridization using human G-CSF receptor cDNA as the probe. Polymerase chain reaction using oligonucleotides specific for the human CSF3R produced a specifically amplified DNA fragment with DNA from mouse A9 cells that contained human chromosome 1 but not other human chromosomes. Localization of the CSF3R on chromosome 1 was further confirmed by the spot-blot hybridization of sorted human chromosomes.     

A subunit of yeast site-specific endonuclease SceI is a mitochondrial version of the 70-kDa heat shock protein

Endo.SceI of Saccharomyces cerevisiae is a heterodimeric site-specific endonuclease, which is distinguishable from prokaryotic restriction endonucleases in the mode of recognition of its cleavage site. We have used monoclonal antibodies specific to the larger subunit (75 kDa) of Endo.SceI to isolate the gene for the subunit (ENS1) from S. cerevisiae. Unexpectedly, ENS1 was found to encode a 70-kDa heat shock protein-related polypeptide and to be identical to recently cloned SSC1. Subcellular fractionation experiments on yeast cells revealed that the primary target site of the larger subunit is mitochondria, where almost all the Endo.SceI activity is localized. Molecular genetic analysis of ENS1 demonstrated its indispensability for growth and the requirement of a high level of its expression at the sporulation and germination stages. The data suggest that ENS1 plays an important role, especially at these differentiation stages.     

Auxin-induced changes in the cell wall structure: Changes in the sugar compositions, intrinsic viscosity and molecular weight distributions of matrix polysaccharides of the epicotyl cell wall of Vigna angularis

Rapid effects of indole-3-acetic acid (IAA) on the mechanical properties of cell wall, and sugar compositions, intrinsic viscosity and molecular weight distribution of cell wall polysaccharides were investigated with excised epicotyl segments of Vigna angularis Ohwi et Ohashi cv. Takara.

• 1 IAA caused cell wall loosening as studied by stress-relaxation analysis within 15 min after the IAA application.
• 2 IAA stimulated the decrease in the content of arabinose and galactose in the hemicellulose 1 h after its application. The amounts of other component sugars in the cell wall polysaccharides remained constant during the IAA-induced segment growth.
• 3 The intrinsic viscocity of the pectin increased as early as 30 min after the IAA application. This effect was not prevented when elongation growth of the segment was osmotically suppressed by 0.15 M mannitol.
• 4 Gel permeation chromatography of the pectin on a Sepharose 4 B column demonstrated that IAA caused increase in the mass-average molecular weight of the pectin. Analysis of the sugar compositions of the pectin eluted from the Sepharose 4 B column indicated that IAA increased the molecular weight of the polysaccharides composed of uronic acid, galactose, rhamnose and arabinose. This effect became apparent within 30 min after the IAA application. Furthermore, IAA increased the molecular weight of the pectin when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.
• 5 Hemicellulose of the cell wall chromatographed on a Sepharose CL-4 B column. Analysis of the neutral sugar compositions and the iodine staining property (specific for xyloglucans) of the polysaccharide solution eluted from the column indicated that the hemicellulose consisted of xyloglucans, arabinogalactans and polysaccharides composed of xylose and/or mannose. IAA caused a decrease in the arabinogalactan content and depolymerization of xyloglucans. These IAA effects became apparent within 30 min after the IAA application. These changes occurred even when elongation growth of the epicotyl segments was osmotically suppressed by 0.15 M mannitol.

Polymerization of the pectin, degradation of arabinogalactans and depolymerization of xyloglucans appear to be involved in the mechanism by which IAA induces cell wall loosening and therefore extension growth of cells.     

A Study on the Mechanism of Nodding Initiation of the Flower Stalk in a Poppy, Papaver Rhoeas L.

A study was made to find whether the nodding of the flower stalkin a poppy, Papaver Rhoeas L., immediately after its formationwas triggered by the weight of its flower bud or by positivegeoreaction and the following results were obtained.

1. The direction of the nodding was mostly toward the inclinedside of the stalk, which was opposite the leaf, for apical flowerbuds.
2. If the weight of the flower bud at stage 1 was cancelledbyapplying a load equivalent to the bud weight, the noddingofthe stalk was not initiated.
3. The stalk at stages 1 and2 and the upper part of the stalk(bending zone), as comparedwith the basal part, at later stageswere highly deformableaccording to measurements by the bendingmethod.
4. The cellwall of highly deformable stalks was rich in hemicellulosesand that of the basal part was abundant in pectic substances.

From these results, we concluded that the initiation of thenodding in the flower stalk was caused by the weight of theflower bud and positive geotropic reaction was probably notinvolved. (Received December 22, 1980; Accepted January 26, 1981)     

Polymerization of deoxyhemoglobin CHarlem (β6 Glu → Val, β73 Asp → Asn): The effect of β73 asparagine on the gelation and crystallization of hemoglobin

The kinetics of aggregation and the solubility of deoxy Hb2 C_Harlem (α₂β₂ 6 Val, 73 Asn) in concentrated phosphate buffers were studied in comparison with those of deoxy Hb S and deoxy Hb A. Deoxy Hb C_Harlem aggregated with a clear exhibition of a delay time. The length of the delay and aggregation times and the degree of the aggregation depended upon the initial hemoglobin concentration.The initial hemoglobin concentration required for the aggregation of deoxy Hb C_Harlem was approximately 200% of its solubility, a value much higher than that required for the aggregation of deoxy Hb S (120%). With the same hemoglobin concentration, the delay time for the aggregation of deoxy Hb C_Harlem was approximately 100 times longer than that of deoxy Hb S. The logarithmic plotting of the delay time versus hemoglobin concentration in 1.8 m-phosphate buffer (pH 7.4) showed linear lines with a slope (n) of 4.0 for deoxy Hb C_Harlem. In contrast to the results for the aggregation of deoxy Hb S, n values for deoxy Hb C_Harlem were unchanged with phosphate concentrations varying from 1.2 m to 2.0 m. The solubilities of deoxy Hb S and deoxy Hb C_Harlem were increased exponentially by lowering the pH of the medium, with the increase being more conspicuous for Hb C_Harlem. The gels (or aggregates) of Hb C_Harlem were converted to crystals at a rate much faster than were those of Hb A and Hb S. The kinetics for gelation and crystallization of deoxy Hb C_Harlem can be explained by the following scheme, where nuclei G and nuclei C are formed before gelation and crystallization, respectively. Monomenc deoxy Hb

The hemoglobin concentration required for the crystallization of deoxy Hb C_Harlem was about ten times lower than that required for deoxy Hb A. The solubility of deoxy Hb C_Harlem after aggregation was about twice that of deoxy Hb S, suggesting that the substitution of Asn for Asp at the β73 residue inhibits the formation of nuclei G and accelerates the formation of nuclei C.     

The concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.