Packing of the 30 nm chromatin fiber in the human metaphase chromosome

The human genetic material is packed hierarchically within the metaphase chromosome: the DNA moleculet together with histone proteins form 11 nm diameter nucleosomes, which are then ordered into the 30 nm thick chromatin fiber. Little is known about the packing of this fiber within the chromosome. We have developed a tracking algorithm with which we followed its path within a three-dimensional reconstruction of a human chromosome computed from a series of electron micrographie projections. Fiber segments were seen to form loops of 100–350 nm diameter. Our observations indicate that these loops — which themselves show no preferred orientation — are organised into regions of roughly 200 nm axial extent.     

CAM-idling in Hoya carnosa (Asclepiadaceae)

In the leaf succulent Asclepiad Hoya carnosa (L.) R. Br., CAM photosynthesis occurred under well-watered conditions, as characterized by diurnal gas exchange and changes in titratable acidity. Following 10–12 days of severe water stress, the plants shifted from CAM to a modified CAM-idling mode of metabolism. CAM-idling was characterized by complete or almost complete stomatal closure accompanied by CAM-like diurnal changes in titratable acidity. H. carnosa plants maintained this CAM-idling mode of photosynthesis for at least 8 weeks. Upon reirrigation, the plants returned to the original CAM mode within 1 week. These results suggested that CAM-idling is a reversible, intermediate form of sustained metabolism which enables plant survival under conditions of extended drought.This work was supported in part by NSF Grant PCM 8200366 and in part by the Science and Education Administration of the United States Department of Agriculture under Competitive Grant 5901-0420-8-0018-0.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Short-term influence of nitrate on acetylene reduction, net photosynthesis and nodule respiration in black alder (alnus glutinosa L. gaertn.) seedlings

The use of nitrogen-fixing trees such as black alder (Alnus glutinosa L. Gaertn.) as forest silvicultural tools has recently been recognized. The potential benefit of black alder in silvicultural practices may be reduced by nitrate fertilization. Fifteen-month-old, nodulated, black alder rooted cuttings were fertilized for 6 days with 0, 7.5 or 15 mM NO₃ to determine the influence of nitrate on acetylene reduction, nodule respiration and net photosynthesis. Acetylene reduction, net photosynthesis and nodule respiration were measured on the second, fourth and sixth days of nitrate application. Nitrate treatment significantly reduced acetylene reduction and nodule respiration by day 4. Acetylene reduction was 75% lower and nodule respiration 36% lower for the 15 mM NO₃ treatment when compared to that of the control treatment. By day 6, net photosynthesis and nodule respiration were significantly reduced by 29 and 59%, respectively, for seedlings treated with 15 mM NO₃. This study suggests that nitrate fertilization has a profound influence on nitrogenase activity and that nitrogen-fixing tree species may respond to nitrate fertilization by shifting photosynthetic rates.     

Lipid composition and food quality of some freshwater phytoplankton for cladoceran zooplankters

The nutritional value of several planktonic algae was testedby means of feeding trials with three cladoceran zooplankters.The algae were monocultures and included two blue-greens, fourgreens and four flagellates with a size range of 5–48µm. The specific growth rates of the zooplankters werechosen as the measure of the nutritional value of the algae.The three cladocerans showed large differences in growth ratein the different algae, but the two cryptomonads were withoutdoubt best suited as food for all. The fatty acid compositionfor the cryptomonads were different from the other algae. Theycontained high percentages of the polyunsaturated fatty acids20:5æ3 (EPA) and 22:6æ3 (DHA), which also are commonin fish. It is suggested that the lipid composition is a probablefactor determining the nutritional quality of the algae.     

Activation of Protein Kinase C Augments Peptide Release from Rat Sensory Neurons

Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 n M phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4α-PDBu analogue at 100 n M had no effect on release. Furthermore, 100 n M bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 n M or 1-oleoyl-2-acetyl- sn -glycerol at 50 µ M did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 n M staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.     

Comparing the spectroscopic and electrochemical properties of ruthenium and osmium complexes of the tridentate polyazine ligands 2,2′:6′,2″-terpyridine and 2,3,5,6-tetrakis(2-pyridyl)pyrazine

A number of osmium and ruthenium complexes of the tridentate ligands 2,2′:6′,2″-terpyridine (tpy) and 2,3,5,6-tetrakis(2-pyridyl)pyrazine (tpp) have been prepared and characterized by our laboratory. All these complexes possess metal based oxidations and ligand based reductions localized on each polyazine ligand. Polymetallic complexes bridged by the tpp ligand exhibit two sequential tpp based reductions prior to the reduction of other polyazine ligands in these complexes. The spectroscopy of these complexes is dominated by ligand based π-π^* transitions in the ultraviolet and MLCT (metal-to-ligand charge transfer) bands terminating on each polyzine ligand in the visible. For the complexes reported herein the lowest lying optical transitionis a M → BL CT band. For most of the complexes reported, occupation of this excited state gives rise to an observable emission at room temperature. For ruthenium complexes of these tridentate ligands, the presence of a low-lying LF state shortens the excited state lifetimes of these chromophores. This gives rise to ruthenium complexes that display shorter excited state lifetimes than the analogous osmium based systems. Incorporation of tpp based chromophores into polymetallic frameworks leads to the production of bimetallic species with long-lived excited states, 100 ns at room temperature. This makes these chromophores good candidates for the development of stereochemically defined supramolecular complexes. It is possible to measure an electrochemical HOMO-LUMO energy gap and a correlation between this electrochemically measured energy gap and the spectroscopic energy associated with this HOMO→LUMO transition are reported herein (HOMO== highest occupied molecular orbital, LUMO = lowest unoccupied molecular orbital).     

Rapid Stimulation of EAAC1-Mediated Na+-Dependent l-Glutamate Transport Activity in C6 Glioma Cells by Phorbol Ester

Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na⁺-dependent l -[³H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[³H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC₅₀ value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in V_max with no change in K_m. PMA also increased the transport of the nonmetabolizable analogue, d -[³H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na⁺-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[³H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca²⁺ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na⁺/H⁺ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na⁺-dependent l -[³H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.     

Duplication of the PMP22 gene in 17p partial trisomy patients with Charcot-Marie-Tooth type-1A neuropathy

Autosomal dominant Charcot-Marie-Tooth type-1A neuropathy (CMT1A) is a demyelinating peripheral nerve disorder that is commonly associated with a submicroscopic tandem DNA duplication of a 1.5-Mb region of 17p11.2p12 that contains the peripheral myelin gene PMP22. Clinical features of CMT1A include progressive distal muscle atrophy and weakness, foot and hand deformities, gait abnormalities, absent reflexes, and the completely penetrant electrophysiologic phenotype of symmetric reductions in motor nerve conduction velocities (NCVs). Molecular and fluorescence in situ hybridization (FISH) analyses were performed to determine the duplication status of the PMP22 gene in four patients with rare cytogenetic duplications of 17p. Neuropathologic features of CMT1A were seen in two of these four patients, in addition to the complex phenotype associated with 17p partial trisomy. Our findings show that the CMT1A phenotype of reduced NCV is specifically associated with PMP22 gene duplication, thus providing further support for the PMP22 gene dosage mechanism for CMT1A. Received: 3 May 1995 / Revised: 1 August 1995