Possible evidence for novel metabolism of nitrogen gas by a green alga

We report the isolation of a cukaryotic green alga ( Chlorella , strain WPI-2) which accumulates large stores of nitrogen (N) during growth in N-free medium and seems to incorporate¹⁴N₂, yet does not reduce acetylene to ethylene. Total N accumulation during growth on N-free medium and in gases free of combined N was measured by three methods: Kjeldahl, oxidative pyrolysis via chemiluminescence (Antek N analyzer), and Dumas (Coleman N analyzer). Increases in N ranging from 22–64%± 1% were observed. Isotope dilution studies using cells labelled with ¹⁵NO ₃- and then shifted to ¹⁴N₂ in N-free medium showed dilution of the ¹⁵N isotope by ¹⁴N from 5.67 to 5.32%± 0.05%. Using a variety of conditions, we were unable to demonstrate the reduction of acctylene to ethylene by WPI-2, although diazotrophic cyanobacteria gave positive results. Although the data on WPI-2 are not conclusive in establishing this alga as a diazotroph, the data do suggest that within the Chlorophyceae there may exist a novel form of nitrogen gas metabolism.     

The stereochemical course of phospho transfer catalyzed by adenylosuccinate synthetase. A reaction pathway via a phosphorylated intermediate with net inversion

The stereochemical course of phospho transfer in the reaction catalyzed by adenylosuccinate synthetase from rat muscle has been determined with chiral [gamma-17O,18O]GTP gamma S as a substrate. The stereochemical configuration of the product, inorganic thiophosphate, was determined by 31P NMR after the compound was stereospecifically incorporated into ATP beta S. The reaction goes with net inversion of configuration, which is the course for a single phospho transfer, even though 6-phospho-IMP is probably an intermediate on the normal reaction pathway (Liebermann, I. (1956) J. Biol. Chem. 223, 327-339). The breakdown of this intermediate goes by C-O bond cleavage and so is not a true phospho transfer step. Thus, inversion of configuration during the course of this ligase reaction is consistent with a single phospho transfer step in the overall reaction, the formation of the phosphorylated intermediate.     

Relationships between macroalgal functional form groups and substrata stability in a subtropical rocky-intertidal system

The general hypothesis that morphological, physiological, and ecological adaptations of macro algal functional-form groups can be related to the level of disturbance encountered in a natural environment was examined. Two articulated calcareous coralline algae (Amphiroa van-bosseae Lemoine, 24% cover and Corallina frondescens Post. & Rupr. 20%) and one non-articulated coralline alga (Lithophyllum sp., 16%), all late-successional predation-tolerant strategists, comprise most of the community cover on stable bedrock substrata at Punta Las Cuevitas, Sonora, Mexico. Conversely, Ulva rigida C. ag. (26% cover) and a ralfsioid crust (23%), shows to be early-successional opportunistic strategists, cover more of the disturbed boulder habitat. Porolithon sonorense Daws., a stress-tolerant strategist, is uniquely abundant on both substratum types (13% cover on boulders, 10% on bedrock). The sheet-like and filamentous algae, prevalent in the temporally unstable habitat, generally show greater productivity (>2×) than the thicker and calcareous forms conspicuous in the more constant environment. It appears that selection for delicate thalli with high productivities, as well as selection for tougher morphologies having lower photosynthetic rates due to greater proportions of structural tissues, are widespread, divergent evolutionary forces among marine algae. Experiments with captive sea urchins (Echinometra vanbrunti Agassiz), in conjunction with fish-preference data published for some of the same algae studied here, offer strong support for the functional-form model. Parrotfishes, rudderfishes, surgeonfishes, damselfishes and E. vanbrunti, in the Gulf of California, preferentially feed on delicate, early-successional, sheet-like, and filamentous algae, while rejecting or ignoring the more structured, late-successional and calcareous algae. There is no significant (P > 0.05) gradation in calorific content between the first four of the six functional groups (i.e., Sheet-, Filamentous-, Coarsely Branched- and Thick Leathery-Groups), but the mean value for these fleshy forms (2.6 kcal · g ash-free dry wt^?1) is significantly greater than that for the last two groups (0.3 kcal, Jointed Calcareous- and Crustose-Groups). The approach used in this study demonstrates a realistic technique for predicting macrophyte community composition from knowledge of the disturbance levels in a given habitat or the reverse. The form group-disturbance relationship has important implications for future biological monitoring of rocky-inter-tidal and subtidal systems.     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.     

Immunocytochemistry of magnocellular neurons of supraoptic and paraventricular nuclei of normal and Brattleboro rats with vasopressin anti-idiotype antibody

Summary A vasopressin anti-idiotype antibody was generated by immunization with purified IgG of a primary vasopressin antiserum. The anti-idiotype antibody immunostained neurons in the supraoptic and paraventricular nuclei of the hypothalamus of normal and Brattleboro rats. The distribution of immunostained perikarya in these hypothalamic nuclei together with the staining of fibers in median eminence and neural lobe was similar to that observed in normal rats with anti-vasopressin and suggests strongly that vasopressinergic neurons are being stained. Absorption studies with vasopressin and a vasopressin-binding receptor protein further indicate that a receptor associated with vasopressinergic neurons is recognized by the anti-idiotype antibody.Supported by NIH grants ES03239, NS18626 and NSF grant BNS-8310914. D.T.P. is the receipient of RCDA award NS00869     

A model of proliferating cell populations with inherited cycle length

A mathematical model of cell population growth introduced by J. L. Lebowitz and S. I. Rubinow is analyzed. Individual cells are distinguished by age and cell cycle length. The cell cycle length is viewed as an inherited property determined at birth. The density of the population satisfies a first order linear partial differential equation with initial and boundary conditions. The boundary condition models the process of cell division of mother cells and the inheritance of cycle length by daughter cells. The mathematical analysis of the model employs the theory of operator semigroups and the spectral theory of linear operators. It is proved that the solutions exhibit the property of asynchronous exponential growth.     

Metastatic phenotype of human prostate tumor cells in athymic nude mice: Alteration by exposure to ethyl methanesulfonate and “reversion” by 5-azacytidine

Summary The human prostate tumor subline 1-LN-PC-3-1A (1-LN) is reproducibly metastatic in adult athymic nude mice. Cells surviving a brief in vitro exposure to ethyl methanesulfonate (EMS) exhibited a profound decrease in capacity for experimental lung metastasis in nude mice. Thirty days after EMS treatment, 1×10⁶ uncloned EMS-treated 1-LN cells (1-LN-EMS-10) were injected IV into groups of 6 to 8-week-old male athymic nude mice (BALB/cAnBOM). A median of 8.5 colonies/lung was observed among 20 1-LN-EMS-10-injected mice, which was significantly different from the median of 51 colonies/lung produced among 14 1-LN-injected mice (P=0.0002). This altered phenotype remained stable during 150 days of continuous culture. However, the 1-LN-EMS-10 cells were tumorigenic in 10/10 nude mice injected SC. Single lung tumor colonies recovered from 1-LN-EMS-10-injected mice and reinjected IV into nude mice produced medians of 32–63 colonies/lung. The altered metastatic phenotype resulting from treatment of 1-LN with EMS was reversed by exposure to a noncytotoxic dose of 5-azacytidine, but unaffected by a second exposure to EMS. Collectively these data demonstrate that the metastatic phenotype of these human tumor cells in athymic nude mice can be heritably altered by in vitro exposure to EMS and 5-azacytidine. Analysis of the mechanisms underlying these phenotypic changes may provide insight into parts of the complex process of tumor cell evolution.     

Enumeration of chlorine-stressed organisms with acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (AOINT)

Direct microscopic enumeration ofEnterobacter cloacae with the acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride technique (AOINT) was compared with spread plate counts on nonselective media to establish the usefulness of the former technique in the enumeration of chlorine-stressed cells. Results indicate that the techniques are comparable when the organisms are not stressed. However, AOINT is more sensitive than are plate counts in the detection of chlorine-stressed cells.     

Proteoglycan extraction of sized cartilage particles

The relationship between cartilage thickness and proteoglycan extractability was examined. Bovine nasal cartilage slices (20, 100, and 500 micron thicknesses) were extracted with low-ionic-strength buffer and 4 M guanidine hydrochloride. The extractability of proteoglycans with both solutions depended on slice thickness. Thinner slices yielded greater amounts of proteoglycans. Sixty-three percent of the total cartilage uronic acid was extracted from 20-micron cartilage slices with low-ionic-strength buffer while only 7% was extracted for 500-micron slices. Each fivefold increase in cartilage surface area led to a threefold increase in uronic acid extraction with low-ionic-strength buffer. Extraction of proteoglycan aggregates was directly proportional to the cartilage surface area whereas extraction of non-aggregated proteoglycans, per surface area, increased with increasing cartilage thickness. These data are consistent with the hypothesis that proteoglycan aggregates are extracted mainly from the cartilage surface while non-aggregated proteoglycans diffuse from deep within the cartilage. Extraction with low-ionic-strength buffer occurred in two phases. There was an initial rapid loss of proteoglycans in which 1/3 to 1/2 of all proteoglycans eluting over 6 days were extracted during the first 30 min. Subsequent extraction was much slower with decreasing amounts extracted on each consecutive day. The initial rapid loss of proteoglycans was probably due to the steep osmotic-pressure gradient existing when the cartilage was placed in the low-ionic-strength buffer.