RECONSTRUCTIONS OF THE GRANA-FRETWORK SYSTEM OF A CHLOROPLAST

The grana-fretwork system was reconstructed from serial sections representing 0.5 μm slice, in profile view, from the midregion of a chloroplast of Nicotiana tabacum. Reconstructions show grana in multiple strata. The fretwork integrates the grana in all three dimensions. Large grana are visualized as complex structures consisting of two or more eccentrically stacked solids that vary in height and diameter. The close spacing and overlapping positions of grana in the plastid explain why phase microscopy cannot be used to demonstrate adequately the numerous strata of grana within a chloroplast. The grana are not far enough apart to allow for successful optical sectioning of a plastid in face view. In profile view no distinct grana can be resolved because the grana-fretwork system has the aspect of a “honeycomb.” Thus, observations with a light microscope are not adequate to determine the arrangement of grana in a chloroplast, and the recently-proposed model showing all grana of a plastid to be arranged in a single spiral ribbon must be rejected as incompatible with properties of real plastids.     

Intramolecular base composition heterogeneity of human DNA.

The intramolecular base composition heterogeneity of human DNA has been investigated by electron microscopic observations of partially denatured structures and by equilibrium solution thermal denaturation techniques. DNA sequences having an average length of less than 2000 base pairs are found to be heterogeneous in base composition. These heterogeneous sequences occupy a minimum of 67 to 81% of the human genome.     

The presence of actin in nuclei: a critical appraisal.

To assess the significance of actin associations with nuclei, we have examined Amoeba proteus nuclei for the presence of labeled actin under a variety of circumstances without (in most instances) isolating nuclei or breaking up cytoplasms prior to the extraction of proteins.We first established that: the 42,000 dalton proteins (presumed to be actin) present in cytoplasm and non-isolated nuclei are identical electrophoretically; the putative actin of amebas has the same size and almost the same isoelectric point as rat muscle actin; and the peptide “fingerprints” of putative ameba actin and rat actin are very similar after tryptic digestion. We therefore concluded that the 42,000 dalton protein of ameba is actin.We determined that: the concentrations of actin in the cytoplasm and nucleus of amebas are the same; actin is readily lost from nuclei that are released from lysed cells; shortly after a ³⁵S-labeled nucleus is transplanted into unlabeled cytoplasm, or an unlabeled nucleus is transplanted into ³⁵S-labeled cytoplasm, the concentration of ³⁵S-actin in nucleus and cytoplasm is the same; and when cells containing ³⁵S-actin are subjected to long chase periods on unlabeled food, the concentrations of ³⁵S-actin in nucleus and cytoplasm fall in parallel. These observations taken together suggest that actin is not tightly associated with nuclei. Rather, actin may associate with nuclei for the trivial reason that the nuclear envelope is no barrier to free movement of that protein between the two compartments.We conclude that the mere presence of actin in nuclei is insufficient grounds for assuming that it has any role in nuclear functions, such as, for example, chromosome condensation.     

Kinetics and Extent of Mineralization of Organic Chemicals at Trace Levels in Freshwater and Sewage

A sensitive and rapid method was developed to measure the mineralization of ¹⁴C-labeled organic compounds at picogram-per-milliliter or lower levels in samples of natural waters and sewage. Mineralization was considered to be equivalent to the loss of radioactivity from solutions. From 93 to 98% of benzoate, benzylamine, aniline, phenol, and 2,4-dichlorophenoxyacetate at one or more concentrations below 300 ng/ml was mineralized in samples of lake waters and sewage, indicating little or no incorporation of carbon into microbial cells. Assimilation of ¹⁴C by the cells mineralizing benzylamine in lake water was not detected. Mineralization in lake waters was linear with time for aniline at 5.7 pg to 500 ng/ml, benzylamine at 310 ng/ml, phenol at 102 fg to 10 mg/ml, 2,4-dichlorophenoxyacetate at 1.5 pg/ml, and di-(2-ethylhexyl) phthalate at 21 pg to 200 ng/ml, but it was exponential at several p-nitrophenol concentrations. The rate of mineralization of 50 and 500 ng of aniline per ml and 200 pg and 2.0 ng of the phthalate per ml increased with time in lake waters. The phthalate and 2,4-dichlorophenoxyacetate were mineralized in samples from a eutrophic but not an oligotrophic lake. Addition to eutrophic lake water of a benzoate-utilizing bacterium did not increase the rate of benzoate mineralization at 34 and 350 pg/ml but did so at 5 and 50 ng/ml. Glucose and phenol reduced the percentage of p-nitrophenol mineralized at p-nitrophenol concentrations of 200 ng/ml but not at 22.6 pg/ml and inhibited the rates of mineralization at both concentrations. These results show that the kinetics of mineralization, the capacity of the aquatic community to assimilate carbon from the substrate or the extent of assimilation, and the sensitivity of the mineralizing populations to organic compounds are different at trace levels than at higher concentrations of organic compounds.     

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.     

Regulation of agonist-induced prostaglandin E1 versus prostaglandin E2 production. A mass analysis.

Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), derived by enzymatic oxidation of cellular dihomogammalinolenic acid (DHLA) and arachidonic acid (AA), respectively, have diverse and, at times, distinct biological actions. It has been suggested that PGE1 specifically inhibits a variety of inflammatory processes, and, in light of the potential therapeutic benefit of PGE1 and its fatty acid precursor in inflammatory disorders, there is growing interest in the biochemical mechanisms which determine the balance between PGE1 and PGE2 synthesis. Metabolic studies in this area have been hampered by the difficulties in measuring the extremely small masses of these prostaglandins which are generated in cell culture systems. We studied the regulation of PGE1 versus PGE2 synthesis using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Because EFD-1 cells contain no endogenous AA or DHLA, we were able to replete the cells with AA and DHLA of known specific activities; thus, the mass of both cellular AA and DHLA, and synthesized PGE1 and PGE2, could be accurately determined. The major finding of this study is that production of PGE2 was highly favored over production of PGE1 due to preferential incorporation of AA versus DHLA into, and release from, the total cellular phospholipid pool. Further, we correlated the selective release of AA versus DHLA from total cellular phospholipids with the selective incorporation of AA versus DHLA into specific phospholipid pools. In addition, we showed that conversion of DHLA to AA by delta 5 desaturase was enhanced by increasing the cellular mass of n-6 fatty acids and by increasing the cell proliferative activity. Together, these results indicate that the relative abundance of PGE2 versus PGE1 in vivo is not merely a function of the relative abundance of AA versus DHLA in tissues, but also relates to markedly different cellular metabolism of these two fatty acids.     

Crystal structure of recombinant rabbit interferon-gamma at 2.7-A resolution

The crystal structure of recombinant rabbit interferon-gamma was solved by the multiple isomorphous replacement technique at 2.7-A resolution and refined to a crystallographic R-factor of 26.2%. The interferon crystallizes with one-half of the functional dimer in the asymmetric unit, with the two polypeptide chains of the dimer related by a crystallographic 2-fold symmetry axis. The structure is predominantly alpha-helical with extensive interdigitation of the alpha-helical segments of the two polypeptide chains.