Secondary plasmodesmata formation in the minor-vein phloem of Cucumis melo L. and Cucurbita pepo L.

Numerous branched plasmodesmata (pd) are present between bundle-sheath cells (BSCs) and specialized companion cells known as intermediary cells (ICs) in the minor-vein phloem of melon (Cucumis melo L.) and squash (Cucurbita pepo L.). These pd were found to be secondary, i.e., they form across existing walls. Sink, sink-source transition, and source tissues were sampled from developing and mature leaves. In sink tissue, IC precursors divide to produce the two to four ICs and associated sieve elements which are present by the time of the sink-source transition. Plasmodesmata along the interface between the IC precursor and adjacent BSCs in sink tissue are unbranched and few in number. Before the leaf tissue undergoes the sink-source transition, the number of pd channels (individual branches of pd) becomes more numerous. This increase in number of pd channels occurs at least in part and perhaps entirely by branching, resulting in more channels on the IC-side than on the BSC-side. In melon there is a 12-fold increase in the number of pd channels within the IC-side of the interface and a corresponding 9-fold increase in pd channels within the BSC-side. Thus, secondary pd form by the time of the sink-source transition and may be involved in phloem loading and photoassimilate export. The system described is well-defined and amenable to experimental manipulation: secondary pd form in large numbers, at a particular interface, over a short period of time, and in a highly predictable manner.Abbreviations BSC bundle-sheath cell - DAP days after planting - IC intermediary cell - LPI leaf plastochron index - pd plasmodesmata - PI plastochron interval We thank Edith Haritatos, Rich Medville, Esther Gowan, and Nancy Dussault for expert technical assistance. This research was supported by an NSF/DOE/USDA Cornell Plant Science Center fellowship (G.M.V.), Natural Sciences and Engineering Research Council Grant GP0138401 and Université de Montréal, Fonds internes de recherche (D.U.B.), and NSF grant IBN-9419703 (R.T.).     

Endogenous opiates: 1994

This article is the 17th installment of our annual review of research concerning the opiate system. It includes papers published during 1994 involving the behavioral, nonanalgesic, effects of the endogenous opiate peptides. The specific topics covered this year include stress; tolerance and dependence; eating; drinking; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurological disorders; electrical-related activity; general activity and locomotion; sex, pregnancy, and development; immunological responses; and other behaviors.     

Gibberellic acid-induced cell elongation in cotton suspension cultures

Gibberellic acid (GA₃) causes cell elongation in cotton suspension cultures derived from cotton ovule callus tissue of both auxin-dependent and-independent lines. Cell elongation was more pronounced in auxin-dependent cultures. Cells were cultured for a period of 14 days but differences in cell lengths could be detected after 6 days in culture. Cell elongation took place in cultures in which GA₃ was present throughout the culture period or only for the first 3 days. Auxins and cytokinin alone or in the presence of GA₃ did not promote cotton cell elongation above the value for the treatment with GA₃ alone.     

Ribosomal Resistance to Streptomycin and Spectinomycin in Neisseria gonorrhoeae

A cell-free protein synthesizing system was used to study the mechanism of resistance to streptomycin (Str) and spectinomycin (Spc) in laboratory mutants and clinical isolates of Neisseria gonorrhoeae. The 70S ribosomes from sensitive strains were sensitive to the effects of Str and Spc on synthesis directed by several synthetic polynucleotide messengers, whereas 70S ribosomes from resistant strains were resistant to these same effects. In each case, the alteration was localized to the 30S ribosomal subunit by studying antibiotic sensitivities of hybrid 70S ribosomes formed by combining subunits from sensitive and resistant strains. No evidence was found for streptomycin- or spectinomycin-inactivating enzymes.     

Ester Synthesis by Zoogloea ramigera 115 Grown in the Presence of Ethanol

The ester ethyl butyrate is produced by Zoogloea ramigera 115, a bacterium isolated from an aerobic waste treatment plant, when ethanol is present in culture media. The cells appear to produce butyric acid which is then esterified with residual ethanol in the culture medium.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Fine scale interspersion of conserved sequences in the pea and corn chloroplast genomes

Summary A comparison is made of the chloroplast genomes of two divergent higher plants, pea and corn. Reassociation kinetics analysis shows that only 33–34% of the chloroplast DNA (ct DNA) sequences are conserved in these two plants, which is equal to about 43 kilobases (kb). The restriction enzyme patterns produced by Eco RI, Bam HI, and Sal I are different for each ct DNA, as expected from the low level of homology. The total length of cross-reacting Eco RI fragments, assessed by blot hybridization methods, exceeds the reassociation kinetics estimate by at least 20 kb. An electron microscopic analysis of ct DNA heteroduplexes shows that the conserved regions are surprisingly short, and consequently, they are interspersed with divergent DNA. Fifty percent of the conserved regions are less than 550 bases; 10 sites are less than 150 bases. The median length of a heterologous region is 250 bases. The heteroduplexes fall into 4 classes, established by the position and size of the conserved and divergent regions, totaling 61 kb. One class has been identified as the ribosomal gene region: the corn Eco RI fragment, Eco RI A, which codes for the 16S and 23S cistrons (Bedbrook and Bogorad 1976), was reassociated with total pea ct DNA, and the products analyzed by electron microscopy. Only one pattern of heteroduplexes was observed. A stretch of almost completely conserved DNA, equivalent to 6.9 kb, extends from the 16S gene through the 23S gene, and therefore, includes the transcribed spacer separating these two cistrons. Heterologous regions occur immediately outside of the 5 and 3 ends of the 16S and 23S genes, respectively. A set of the 16S and 23S genes contribute about 4%, and the spacer 1.6%, to the level of sequence homology in each genome.G.K.L. was supported by a National Research Service Award from the National Institutes of Health (GM 07270). This work was also supported by National Institutes of Health Grant GM 22870, and a Grant-in-Aid of Research to G.K.L. from Sigma Xi, The Scientific Research Society     

Allelopathic compounds,thelypterin A and B in the fern Thelypteris normalis

Summary The growth of Thelypteris normalis (C. Chr.) Moxley gametophytes is inhibited under T. normalis sporophytes. Competition for minerals, light, change in pH, or microbial inhibitors were experimentally eliminated as causes of the inhibition. This is the first demonstration of allelopathy between a sporophyte and gametophyte in a fern. Two inhibitors, thelypterin A and B, which were released from the roots of the Thelypteris sporophyte, were isolated and a bioassay for the inhibitors was devised. Thelypterin A gave an Ehrlich-positive reaction indicative of secondary aromatic amines and an ultraviolet absorption spectrum indicative of a heterocyclic ring. The inhibitors affected the growth of Thelypteris, Pteris and Phlebodium gametophytes.     

Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase. Modulation of activity by multiple factors.

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.