Electron microprobe analysis of juvenile walleye pollock,Theragra chalcogramma,otoliths from Alaska: a pilot stock separation study

Synopsis The incorporation of dissolved oceanic constituents in the otoliths of fish has potential as a chemical tracer for reconstructing the early life history of marine fish. Wavelength dispersive spectrometers on an electron microprobe were used to measure Na, Mg, P, S, Cl, K, Ca, and Sr concentrations on the outer margins of 57 juvenile walleye pollock, Theragra chalcogramma, otoliths from five locations in the Gulf of Alaska and Bering Sea. Discriminant analyses that used various combinations of Na, P, K, Sr, and fish standard length and/or age showed that 60–80% of the samples could be assigned to the correct capture locality. While the concentrations of some of the measured elements correlated with standard length or age of the fish, there are measurable differences among localities when concentrations are length or age corrected, mainly due to differences in Na and K concentrations. Elemental composition of otoliths potentially could be used to assign fish from a mixed stock fishery to original stocks, information that is greatly needed for the effective management of fish stocks.     

Localization of stomatogastric IV neuron cell bodies in lobster brain

Summary The cell bodies of the inferior ventricular nerve (IVN) through-fibers of the lobster stomatogastric nervous system were located using cobalt chloride backfills and intracellular recordings. Following backfills of the IVN, two cell bodies in the supraesophageal ganglion (or brain) were stained with cobalt. These cells, each approximately 30 m in diameter, were located at the base of the IVN, just inside the connective tissue sheath surrounding the brain, and were identifiable on the basis of their close proximity to the IVN.In order to record from the cells, an in vitro preparation was made which included the cell bodies, their axons in the IVN and the stomatogastric nervous system. Intracellular recordings showed that the axons projected to the stomatogastric ganglion and made synaptic connections onto identified neurons. The axon trajectories and synaptic connections correlated with those previously described for the IVN through-fibers using extracellular stimulation and recording techniques.Abbreviations IVN inferior ventricular nerve - SN stomatogastric nerve     

Reciprocal translocation and the Philadelphia chromosome

Summary We examined metaphases from three patients with chronic myeloid leukaemia and a typical Philadelphia chromosome with one chromosome 9 as the recipient to determine whether the 9q+ 22q- translocation is reciprocal. Good quality G-banded photographs of the chromosomes concerned were subjected to light absorption density analysis. This provided enlarged tracings corresponding to the relevant chromosome regions and so facilitated accurate measurement. This technique has unambiguously shown that the typical Philadelphia chromosome results from a reciprocal translocation and that probably no material is gained or lost in the exchange. Furthermore, in a total of six patients for whom sequential G and C banding was performed, the chromosome 9 with the largest block of centromeric heterochromatin received the translocated material. We offer tentative explanations for this curious observation.     

Radical mechanism of aminopyrine oxidation by cumene hydroperoxide catalyzed by purified liver microsomal cytochrome P-450

Under identical experimental conditions, purified preparations of rabbit liver microsomal cytochrome P-450 and beef heart metmyoglobin were equally effective at stimulating the oxidation of aminopyrine to a free radical species by cumene hydroperoxide. Mannitol had no effect on radical levels produced with either hemeprotein-hydroperoxide system; however, specific ligands of the two hemeproteins, substrates of cytochrome P-450, and phospholipid affected the two systems quite differently. Only the metmyo-globindependent oxidation of aminopyrine was significantly inhibited by fluoride and cyanide. Metyrapone, a specific ligand of cytochrome P-450, and benzphetamine, which was N-demethylated by cumene hydroperoxide only in the presence of cytochrome P-450, inhibited only the cytochrome P-450-stimulated oxidation of aminopyrine. Moreover, only with the solubilized liver hemeprotein was aminopyrine radical generation markedly stimulated by phospholipid. Similar properties of aminopyrine N-demethylation and radical formation by the cytochrome P-450-cumene hydroperoxide system have strongly implicated the radical as a requisite intermediate in product formation. Micromolar concentrations of metyrapone caused parallel inhibition, by at least 50%, of both radical generation and formaldehyde production. These results support a radical pathway of N-demethylation proposed for other hemeprotein-hydroperoxide systems (B. W. Griffin and P. L. Ting, 1978, Biochemistry, 17, 2206–2211), in which the substrate undergoes two successive one-electron abstractions, followed by hydrolysis of the iminium cation intermediate. Thus, for this class of substrates, the experimental data are consistent with the oxygen atom of the product arising from H₂O and not directly from the hydroperoxide, which has been previously proposed as a general mechanism for cytochrome P-450 peroxidatic activities.     

Rescue and Transmission of a Replication-Defective Variant of Moloney Murine Leukemia Virus

We have described a clone of mouse cells, termed "8A," which appears to be infected with a replication-defective variant of Moloney murine leukemia virus (MuLV) (Rein et al., J. Virol. 25:146-156, 1978). Clone 8A cells release virus particles which do not form plaques in the standard XC test. However, approximately 10(2) particles per ml of clone 8A supernatant do form plaques in a modified XC test (the "complementation plaque assay"), in which the assay cells are coinfected with the XC-negative, nondefective amphotropic MuLV as well as the test virus. Superinfection of clone 8A cells themselves with amphotropic MuLV results in the production of approximately 10(5), rather than approximately 10(2), particles per ml which register in the complementation plaque assay. This increase is due to the rescue of replication-defective ecotropic MuLV from clone 8A cells by amphotropic MuLV since (i) this ecotropic MuLV can only form XC plaques in cells which are coinfected with amphotropic MuLV; and (ii) it is possible to transmit this defective variant, rescued from superinfected clone 8A cells, to a fresh clone of normal mouse cells. The time course of production of the rescued MuLV particles by superinfected clone 8A cells is virtually identical to that of rescue from these cells of murine sarcoma virus. Amphotropic MuLV superinfection of "NP-N" cells, which contain a "non-plaque-forming" variant of N-tropic MuLV (Hopkins and Jolicoeur, J. Virol. 16:991-999, 1975), also increases the titer of particles registering in the complementation plaque assay; thus, NP-N cells, like clone 8A cells, contain a rescuable defective variant of ecotropic MuLV.     

Teteraene and pentaene leukotrienes: Selective production from murine mastocytomo cells after dietary manipulation

A neoplastic mast cell tumor was grown in mice which had been raised since birth on a diet enriched with eicosapentaenoic acid. Intact harvest mastocytoma cells were stimulated with calcium ionophpore A23187 to produce lipoxygenase products from the polyunsaturated fatty acids liberated from the cellular membranes. Leukotriene B₄, B₅, C₄ and C₅ were isolated and characterized by HPLC retention time, ultraviolet absorption spectrometry and mass spectrometry. The arachidonic acid content of the mast cell tumor lipids was altered from 9.2 to 3.9 mole% while eicosapentaenoic acid increased from 0.5 to 4.5 mole % in response to the fish oil-supplement diet.The relative amount of arachidonic and eicosapentaenoic acids (3.9 and 4.5 mole % respectively) were associated with similar amounts of LTB₄ and LTP₅ synthesized by the cells. These results suggest that the epoxide leukotrine (LTA) derivative can be made efficiently from either arachidonic or eicosapentaenoic acids when both are present in cellular lipids. In contrast, the ratio of LTC₄ to LTC₅ (10 to 1) indicates that the reaction of LTA with glutathione may be critically dependent upon the structure of the unsaturated fatty acid with the ratio of LTC₄/LTB₄ (2.0) more than 10 times greater than that (0.16) for LTC₅/LTP₅.     

Use of hydrogen cyanamide for apple and plum thinning

Effects of various concentrations of Dormex (a.i. 49% hydrogen cyanamide) on fruit thinning of Rome Beauty apple (Malus domestica Borkh.), Friar and Simka plums (Prunus salicina Lindley) were studied. A full bloom application of Dormex at all tested concentrations decreased Rome Beauty apple fruit set and yield, and increased fruit weight. Dormex at 0.25% (v/v) resulted in adequate apple thinning, indicated by production of an optimum fruit weight (320 g). Prebloom and full bloom applications of Dormex at greater than 0.75% reduced plum fruit set and yield in Friar. Full bloom application of Dormex at 0.50% showed a satisfactory fruit set, yield, and fruit weight in Friar plum. Prebloom Dormex application had no significant effect on `Simka' plum fruit set or yield, but full bloom application decreased fruit set and yield.     

Use of a Plasmid DNA Probe To Monitor Populations of Bacillus pumilus Inoculant Strains in Hay

We are evaluating naturally occurring isolates of Bacillus pumilus for use as microbial hay preservatives. Seven isolates of B. pumilus from hay contained a 42-kb cryptic plasmid (pMGD296). We wished to determine whether pMGD296 could be used as a molecular marker to follow populations of these isolates in hay over time. Southern blots and colony blots of 69 isolates of B. pumilus and other Bacillus spp. were probed with ³²P-labeled pMGD296. Twenty-nine probe-positive isolates were identified; of these, 28 contained a plasmid with a restriction profile identical to that of pMGD296. One isolate from untreated hay contained a 40-kb plasmid (pMGD150) that was homologous to pMGD296 but had a different restriction fragment pattern. Regions of homology between the two plasmids were identified by Southern blotting, and a 1.9-kb HindIII-PstI fragment of pMGD296 lacking strong homology to pMGD150 was cloned in pUC18. The cloned fragment hybridized only with isolates containing pMGD296 and was used to estimate populations of these isolates in treated and untreated hay.     

Growth and characterization of human skin epithelial cell cultures

Summary In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epithelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis. Expert technical assistance was provided by Nancy Allen (cell culture); William Towler (electron microscopy); James Malone, Nona Scaife, and Joy M. Nicolet (cytogenetics); R. Thomas Campbell and Dorothy Sarver (photography); and V. L. Angerstein, Susan Ekker, and Arnater Yarbrough (histology). This work was supported by The United Fund Cancer Society of Summit County, the Greater Cleveland Associated Foundation (grant no. 3G3490X1), the National Institute of General Medical Services (grant no. 1 R01 GM 21929-01), and the Charles E. Merrill Trust.