Phenotypic variation during micropropagation of the chimeralRhododendron ‘President Roosevelt’

The putative periclinal chimeraRhododendron xlimbatum President Roosevelt was used to study the origin of shoots in vitro. Genotypic segregation readily occurred in vitro. Numerous phenotypes were observed, although most shoots were either entirely green or maintained the original variegation pattern. Derivatives of the third apical layer were rarely involved in shoot formation. A reversed chimeral form was isolated. Adventitious shoots were usually miniaturized and rapidly proliferating, but axillary shoots had thicker stems, larger leaves and proliferated more slowly. Corolla tissue produced stunted, leafy shoots; no variegated shoots were produced from floret explants. In shoot tip cultures the addition of 40M 2iP without IBA resulted in the greatest number of shoots. Explant choice was the most critical factor for maintenance of foliar variegation.     

Assessment of methods to determine minimal cellulase concentrations for efficient hydrolysis of cellulose

Summary The enzyme loading needed to achieve substrate saturation appeared to be the most economical enzyme concentration to use for hydrolysis, based on percentage hydrolysis. Saturation was reached at 25 filter paper units per gram substrate on Solka Floc BW300, as determined by studying (a) initial adsorption of the cellulase preparation onto the substrate, (b) an actual hydrolysis or (c) a combined hydrolysis and fermentation (CHF) process. Initial adsorption of the cellulases onto the substrate can be used to determine the minimal cellulase requirements for efficient hydrolysis since enzymes initially adsorbed to the substrate have a strong role in governing the overall reaction. Trichoderma harzianum E58 produces high levels of -glucosidase and is able to cause high conversion of Solka Floc BW300 to glucose without the need for exogenous -glucosidase. End-product inhibition of the cellulase and -glucosidase can be more effectively reduced by employing a CHF process than by supplemental -glucosidase.Offprint requests to: C. M. Hogan     

DNA fingerprinting of lactococci and streptococci used in dairy fermentations

Summary A DNA fingerprinting procedure was developed for strains of Lactococcus lactis subsps. lactis and cremoris, biovar. diacetylactis, and Streptococcus salivarius subsp. thermophilus, used in dairy fermentations. Total cellular DNA was extracted and digested with restriction endonucleases, HindIII or HaeIII, followed by separation of the fragments using agarose gel electrophoresis. L. lactis C2 was used as a representative strain for examining the effect of growth phase and cell concentration, cell washing conditions prior to lysis, type and concentration of the enzyme used to digest the cell wall, composition of the lysis buffer, and gel electrophoresis conditions. Following optimization of the fingerprinting procedure, electrophoretic migration of fragments from 23 strains produced reproducible gel patterns. L. lactis subsp. lactis strains ML3 and C2 appeared to be identical when restrricted with either Hind III or HaeIII. Similarly, S. salivarius subsp. thermophilus strains 19987 and 19258, and L. lactis subsp. cremoris strains 134 and C3, appeared to have identical DNA fingerprints following digestion with HindIII. To determine the usefulness of this technique for monitoring population changes during fermentation, various ratios of two closely related strains were inoculated into milk and allowed to grow for 16 h at 32° C. The initial inoculum ratios were determined by standard plate counts, and the final ratio was deterimined by DNA fingerprinting. DNA fingerprinting will be useful in the identification, characterization, and comparison of food fermentation microorganisms.Published as paper No. 17,803 of the contribution series of the Minnesota Agricultural Experiment Station Offprint requests to: S. K. Harlander     

Seed dispersal spectra: a comparison of temperate plant communities

Abstract. We compare the dispersal spectra of diaspores from varied plant communities in Australia, New Zealand, and North America, assigning dispersal mode to each diaspore type on the basis of apparent morphological adaptations. Species with ballistic and external dispersal modes were uncommon in most communities we surveyed. Ant dispersal was also rather uncommon, except in some Australian sclerophyll vegetation types. The frequency of vertebrate dispersal ranged up to 60% of the flora, the highest frequencies occurring in New Zealand forests. Wind dispersal ranged as high as 70% of the flora, with the highest values in Alaska, but usually comprised 10–30% of the flora. Many species in most communities had diaspores with no special morphological device for dispersal. Physiognomically similar vegetation types indifferentbiogeographic regions usually had somewhat dissimilar dispersal spectra. The frequency of dispersal by vertebrates often increased and the frequency of species with no special dispersal device decreased along gradients of increasing vertical diversity of vegetation structure. Elevation and moisture gradients also exhibited shifts in dispersal spectra. Within Australia, vertebrate- and wind-dispersal increased in frequency along a soil-fertility gradient, and dispersal by ants and by no special device decreased. Habitat breadths (across plant communities) and microhabitat breadths (within communities) for species of each major dispersal type did not show consistent differences, in general. Ant-dispersed species often had lower cover-values than other species in several Australian vegetation types. We discuss the ecological bases of these differences in dispersal spectra in terms of the availability of dispersal agents, seed size, and other ecological constraints. Seed size is suggested to be one ecological factor that is probably of general relevance to the evolution of dispersal syndromes.     

MPphiWR-2, a cryptic phage associated withMicromonospora purpurea ATCC 15835

Summary A previously undescribed cryptic phage was found associated withMicromonospora purpurea ATCC 15835.     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

Mechanisms of Injury-Induced Calcium Entry into Peripheral Nerve Myelinated Axons: Role of Reverse Sodium-Calcium Exchange

Abstract: To investigate the route of axonal Ca²⁺ entry during anoxia, electron probe x-ray microanalysis was used to measure elemental composition of anoxic tibial nerve myelinated axons after in vitro experimental procedures that modify transaxolemmal Na⁺ and Ca²⁺ movements. Perfusion of nerve segments with zero-Na⁺/Li⁺-substituted medium and Na⁺ channel blockade by tetrodotoxin (1 µM) prevented anoxia-induced increases in Na and Ca concentrations of axoplasm and mitochondria. Incubation with a zero-Ca²⁺/EGTA perfusate impeded axonal and mitochondrial Ca accumulation during anoxia but did not affect characteristic Na and K responses. Inhibition of Na⁺-Ca²⁺ exchange with bepridil (50 µM) reduced significantly the Ca content of anoxic axons although mitochondrial Ca remained at anoxic levels. Nifedipine (10 µM), an L-type Ca²⁺ channel blocker, did not alter anoxia-induced changes in axonal Na, Ca, and K. Exposure of normoxic control nerves to tetrodotoxin, bepridil, or nifedipine did not affect axonal elemental composition, whereas both zero-Ca²⁺ and zero-Na⁺ solutions altered normal elemental content characteristically and significantly. The findings of this study suggest that during anoxia, Na⁺ enters axons via voltage-gated Na⁺ channels and that subsequent increases in axoplasmic Na⁺ are coupled functionally to extraaxonal Ca²⁺ import. Intracellular Na⁺-dependent, extraaxonal Ca²⁺ entry is consistent with reverse operation of the axolemmal Na⁺-Ca²⁺ exchanger, and we suggest that this mode of Ca²⁺ influx plays a general role in peripheral nerve axon injury.     

Eph Family Receptors and Their Ligands Distribute in Opposing Gradients in the Developing Mouse Retina

The Eph family of receptor tyrosine kinases and their ligands can be divided into two specificity subclasses: the Eck-related receptors and their GPI-anchored ligands, and the Elk-related receptors and their transmembrane ligands. Previous reports demonstrated that Eck- and Elk-related receptors in the retina distribute in high temporal–low nasal and high ventral–low dorsal gradients, respectively. While others have focused on complementary ligand gradients in the retinal axon target, the tectum, we report that ligands from each subclass also distribute in gradients opposing those of their corresponding receptors within the retina itself. Moreover, ligand gradients in the retina precede ganglion cell genesis. These results support an intraretinal role for Eph family members in addition to their previously proposed role in the development of retinotectal topography. The distinct distributions of Eph family members suggest that each subclass specifies positional information along independent retinal axes.     

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     

Opioid receptors: Some perspectives from early studies of their role in normal physiology,stress responsivity,and in specific addictive diseases

The early history of research on the possible existence of specific opioid receptors and on developing a new form of pharmacotherapy for the treatment of heroin addiction in New York City, from 1960–1973, along with the special relationships between two leading scientists conducting these research efforts, Dr. Eric Simon and Dr. Vincent P. Dole Jr., are presented in a historical perspective. The linkage of these early efforts and the subsequent identification and the elucidation of the effects of exogenous opiates acting at specific opiate receptors in human physiology, including some findings from perspective studies of heroin addicts at time of entry to and during methadone maintenance treatment, are presented in the context of the important clues which thereby were provided concerning the possible roles of the endogenous opioids in normal mammalian physiology. From many of these early clinical research findings and studies in animal models, the hypothesis that the endogenous opioids system may play an important role in stress responsivity was formulated along with the related hypothesis, first presented in the early 1970s, that an atypical responsivity to stress and stressors might be involved in the acquisition and persistence of, and relapse to specific addictive diseases, including heroin addiction, cocaine dependency and alcoholism. More recent studies of the possible involvement of the specific opioid receptors in these three addictive diseases—heroin addiction, cocaine addiction and alcoholism—from our laboratory are discussed in a historical perspective of the development of these ideas from the early research findings of not only Dr. Eric Simon, but his numerous colleagues in opioid research in the United States and throughout the world. Special issue dedicated to Dr. Eric J. Simon.