Isolation and characterization of a glycosylated form of beta nerve growth factor in mouse submandibular glands

In the course of characterizing polyclonal antibodies to beta nerve growth factor (NGF) on immunoblot replicas of sodium dodecyl sulfate gels, we observed a protein (designated C protein) migrating as two bands (14.0 and 13.5 kDa) that copurifies with NGF and reacts strongly with its antibodies. The molecule is detectable in the 7 S, beta, and 2.5 S forms of NGF, accounting in the latter two for approximately 2% of total protein. The C protein can be separated from the A and B chains of beta-NGF on acetic acid-urea gels and on two-dimensional gels but not by isoelectric focusing alone. The molecule has been isolated to near purity on reversed-phase high performance liquid chromatography. Amino acid analyses and sequencing through 49 Edman cycles revealed that the protein preparation is composed of the intact and desoctapeptide (des-(1-8] polypeptide chains and suggested a glycosylation site at Asn-45. Following digestion with N-glycanase, the chains migrated on sodium dodecyl sulfate gels identically with the A and B chains of beta-NGF. Although this was accompanied by some degree of proteolytic degradation, the presence of glucosamine (approximately 4 mol/mol of single chain) was confirmed in acid hydrolysates on the amino acid analyzer. No amino sugars were detected in hydrolysates of the A chain nor was galactosamine recovered in either preparation. Glycosylated NGF promotes neuronal growth and survival in a manner indistinguishable from native 2.5 S NGF when tested in the chick sensory ganglion assay and with rat postnatal sympathetic neurons in a dissociated culture cell survival assay or in a compartmentalized culture growth assay. These studies reveal that NGF can be modified by glycosylation in a manner that does not reduce its biological activity.     

Ant predation on an invasive herbivore: can an extrafloral nectar-producing plant provide associational resistance to <Emphasis Type="Italic">Opuntia</Emphasis> individuals?

The legume Chamaecrista fasciculata attracts ants to its extrafloral nectar (EFN) which can lead to reduced herbivory and increased fecundity for the plant. In Florida, Opuntia stricta and O. humifusa, hosts of the invasive moth Cactoblastis cactorum, are often found growing in close association with C. fasciculata. We tested the hypotheses that O. stricta and O. humifusa individuals have higher ant abundance, lower levels of herbivore damage, and increased growth when growing in close association with C. fasciculata compared with individuals not growing near the plant. We also experimentally placed C. cactorum eggsticks and pupae on Opuntia individuals to see if ant predation of these stages occurred, and if so, whether predation rates were higher on individuals growing close to C. fasciculata. Opuntia plants near C. fasciculata were less likely to be attacked by C. cactorum and had higher ant abundance than plants far from C. fasciculata. Field surveys showed that Opuntia plants near C. fasciculata had a lower proportion of cladodes with C. cactorum damage of any type. Proportions of cladodes with damage from five native herbivores were not significantly different between treatments. In addition, Opuntia individuals growing near C. fasciculata added proportionately more pads during the growing season. We found evidence of ant predation on 15.9% of C. cactorum eggsticks and 17.6% of pupae. In August and October of 2008, there was significantly more evidence of predation on eggs and pupae placed on Opuntia individuals near C. fasciculata. No effect of distance to C. fasciculata was seen in November of 2008, potentially because plants were no longer producing EFN at this time. Our finding that Opuntia plants close to C. fasciculata show reduced herbivory from invasive C. cactorum, but not from the native herbivores examined, suggests that patterns of associational resistance may be influenced by the co-evolutionary history of the organisms in question.     

Computational simulation of spontaneous bone straightening in growing children

Periosteal surface pressures have been shown to inhibit bone formation and induce bone resorption, while tensile strains perpendicular to the periosteal surface have been shown to inhibit bone resorption and induce new bone deposition. A new computational model was developed to incorporate these experimental findings into simulations of spontaneous bone straightening in children with congenital posteromedial bowing of the tibia. Three-dimensional finite element models of the periosteum were used to determine the relationships between the defect angle and the distribution of bone surface pressures and strains due to growth-generated tensile strains in the periosteum. These relationships were incorporated into an iterative simulation to model development of a growing, bowed tibia with an initial defect angle of 27°. When periosteal loads were included in the simulation, the defect angle decreased to 10° after 2 years, and the bone straightened by an age of 25 years. When periosteal loads were not included in the simulation, the defect angle decreased to 23° after 2 years, and a defect angle of 9° remained at an age of 25 years. A “modeling drift” bone apposition/resorption pattern appeared only when periosteal loads were included. The results suggest that periosteal pressures and tensile strains induced by bone bowing can accelerate the process of bone straightening and lead to more complete correction of congenital bowing defects. Including the mechanobiological effects of periosteal surface loads in the simulations produced results similar to those seen clinically, with rapid straightening during the first few years of growth.     

Ammonium excretion and glutamate dehydrogenase activity of zooplankton in Great South Bay, New York

In Great South Bay, nanoplankton, (<20 sµm) accountedfor the largest fraction (56%) of zooplankton glutamate dehydrogenase(GDH) activity over a one year period. Microzooplankton (20–200µm) and macrozooplankton (>200 µm) accountedfor 20% and 24%, respectively. Total zooplankton ammonium regenerationin Great South Bay could account for 74% of the ammonium requirementby phytoplankton in winter, but in summer when phytoplanktondemand was greater, and zooplankton population was low, it suppliedless than 5%. This study suggests that the smallest zooplanktonfraction, less than 20 µm, can be the most important asregards nitrogen regeneration in estuarine environments. MacrozooplanktonGDH activity in Great South Bay ranged from 0.18 mg atoms NH⁺₄-Nm^–3 d^–1 in winter to 3.34 mg atoms NH⁺₄-N m^–3d^–1 in spring. Over an annual period, the averaged GDH/excretionratio was 20.4 3.5 (n = 10), and this ratio agrees well withobservations by other investigators. Observed macrozooplanktonexcretion rates showed a strong correlation with the excretionrates indirectly estimated from GDH activities. The GDH/excretionratio seems to vary depending on the internal physiologicalstates of zooplankton as well as food availability.     

Analysis of Amblyomma sculptum haplotypes in an area endemic for Brazilian spotted fever

Amblyomma sculptum (Ixodida: Ixodidae) Berlese, 1888, a member of the Amblyomma cajennense complex, is the major vector of Brazilian spotted fever (BSF) in southeastern Brazil. In this study, the genetic diversity of A. sculptum populations in the state of Rio de Janeiro (RJ), Brazil, was investigated because genetic variability in tick populations may be related to vector competence. Samples of A. sculptum from 19 municipalities in 7 regions of RJ were subjected to DNA extraction, amplification and sequencing of D‐loop, cytochrome oxidase II and 12S rDNA mitochondrial genes. These sequences were used to map the genetic diversity of this tick. Amblyomma sculptum populations are genetically diverse in RJ, especially in the South Centre and Highland regions. Few unique haplotypes were observed in all populations, and the majority of genetic variation found was among ticks within each population. Phylogenetic reconstruction reinforced the assumption that all the haplotypes identified in RJ belong to A. sculptum. However, some RJ haplotypes are closer to A. sculptum from Argentina than to A. sculptum from elsewhere in Brazil. In RJ, A. sculptum has high genetic diversity, although little genetic differentiation. Observations also indicated a high level of gene flow among the studied populations and no evidence of population structure according to region in RJ.     

Action of lead on glutamate-activated chloride currents inHelix pomatia L. neurons

Summary 1. In molluscan neurons glutamate may, on different neurons, evoke either excitation or inhibition. We studied neurons ofHelix pomatia which have hyperpolarizing responses to glutamate and determined the effects of lead on these responses.2. In voltage clamp experiments, the reversal potentials of these glutamate responses indicate that they are due to a conductance increase to chloride ions. Further evidence for this conclusion was obtained by the demonstration that responses to glutamate remained unaffected in experiments with intracellular dialysis with K-free saline in the presence of Na- and K-free extracellular media. In these circumstances, there is effectively no other ion than chloride to carry the current. In isolated neurons the glutamate-evoked chloride current is concentration dependent between 25 and 2500 µM. The current rises over 200 msec and declines in the continued presence of glutamate over a period of about 3 sec.3. Lead (0.5–1.0 µM) potentiated the glutamate-evoked chloride current provided that the channels were not maximally activated. The potentiation was greater if lead was added 30–60 sec before glutamate application.4. These results suggest that potentiation of transmitter-evoked responses by lead must be considered as yet another possible site of action of lead on neurons, and thus this effect must be considered as a part of the mechanism responsible for the neurotoxicity of this heavy metal.     

Inclusion bodies in several species of Ceratium Schrank (Dinophyceae) from the Caribbean Sea examined with DNA-specific staining

Several dinoflagellate species in the genus Ceraiium Schrankwere examined, with the help of a DNA-specific dye, for thepresence of a nucleus, chloroplasts and inclusion bodies. Theabundance of Ceratium in the Caribbean Sea in April 1990 wasbetween 1.3 and 4.9 x 10⁵ cells m^–3 Most Ceratium teresKofoid contained 5–10 small chloroplasts and among 529C.teres cells. 8% possessed an inclusion body. The shape andsize of inclusion bodies varied, and 7% of the inclusions hada DNA-containing particle inside. Similar inclusion bodies werealso observed in Ceratium declinatum f. normale Jörgensen,Ceratium furca (Ehrenberg) Claparede and Lachmann, and Ceratiumfusus (Ehrenberg) Dujardin. Our observations indicated thatinclusion bodies are common in oceanic Ceratium. ¹Present address: Institute of Marine Biology, National TaiwanOcean University, Keelung 20224, Taiwan, ROC