Additive effects of aboveground and belowground herbivores on the dominance of spotted knapweed (Centaurea stoebe)

Spotted knapweed (Centaurea stoebe) is found in over 3 million ha of rangeland and forests across North America, and evidence supporting the use of biological control as a regional method to reduce infestations and their associated impacts remains inconclusive. Several species of insects have been reported to reduce plant densities in some areas; however, rigorous studies that test combinations of these species and the influence of resource availability are lacking. We examined the singular and combined effects of herbivory by a root weevil (Cyphocleonus achates) and a flower head weevil (Larinus minutus) on the growth and flower production of C. stoebe. We also manipulated soil resource fertility as an additional factor that could explain the outcomes of contradictory biological control herbivore effects on C. stoebe. In a greenhouse study, herbivory by C. achates decreased flower production for plants across all resource environments. In a caged common garden study, the negative effects of herbivory also did not interact with soil nutrient status. However, the presence of plant competition further decreased knapweed growth, and the negative effects of concurrent herbivory by C. achates and L. minutus on plant biomass and flower production were additive. Derived within the context of variable levels of soil nutrient availability and competing vegetation, these results support the cumulative stress hypothesis and the contention that combined above- and belowground herbivory can reduce spotted knapweed densities and reduce the ecological and economic impacts of this species in rangelands of western North America.     

Molecular modeling of cardiac glycoside binding by the human sequence monoclonal antibody 1B3

The amino acid sequences of the heavy- and light-chain variable regions of the high-affinity human sequence antidigoxin monoclonal antibody 1B3 (mAb 1B3) were determined, and a structural model for the mAb's variable region was developed by homology modeling techniques. The structural model provided the basis for computationally docking digoxin and eight related cardiac glycosides into the putative binding site of mAb 1B3. Analysis of the consensus binding mode obtained for digoxin showed that the cardenolide moiety of digoxin is deeply embedded in a predominantly hydrophobic, narrow cavity, whereas the terminal, gamma-carbohydrate group is solvent-exposed. The docking results indicated that the primary driving forces for digoxin binding by mAb 1B3 are hydrophobic interactions with the digoxin steroid ring system and hydrogen bonds with the digitoxose groups. The binding model accounts for the experimentally observed variations in mAb 1B3 binding affinity for various structural analogs of digoxin used previously to develop a 3D structure-activity relationship model of drug binding (Farr CD, Tabet MR, Ball WJ Jr, Fishwild DM, Wang X, Nair AC, Welsh WJ. Three-dimensional quantitative structure-activity relationship analysis of ligand binding to human sequence antidigoxin monoclonal antibodies using comparative molecular field analysis. J Med Chem 2002;45:3257-3270). In particular, the hydrogen bond pattern is consistent with the unique sensitivity of mAb 1B3's binding affinity to the number of sugar residues present in a cardiac glycoside. The hydrophobic environment about the steroid moiety of digoxin is compatible with the mAb's reduced affinity for ligands that possess hydrophilic hydroxyl and acetyl group modifications in this region. The model also indicated that most of the amino acid residues in contact with the ligand reside in or about the three complementarity determining regions (CDRs) of the heavy chain and the third CDR of the light chain. A comparison of the 1B3 binding model with the crystal structures of two murine antidigoxin mAbs revealed similar binding patterns used by the three mAbs, such as a high frequency of occurrence of aromatic, hydrophobic residues in the CDRs and a dominant role of the heavy chain CDR3 in antigen binding.     

Polarized secretory trafficking directs cargo for asymmetric dendrite growth and morphogenesis

Proper growth of dendrites is critical to the formation of neuronal circuits, but the cellular machinery that directs the addition of membrane components to generate dendritic architecture remains obscure. Here, we demonstrate that post-Golgi membrane trafficking is polarized toward longer dendrites of hippocampal pyramidal neurons in vitro and toward apical dendrites in vivo. Small Golgi outposts partition selectively into longer dendrites and are excluded from axons. In dendrites, Golgi outposts concentrate at branchpoints where they engage in post-Golgi trafficking. Within the cell body, the Golgi apparatus orients toward the longest dendrite, and this Golgi polarity precedes asymmetric dendrite growth. Manipulations that selectively block post-Golgi trafficking halt dendrite growth in developing neurons and cause a shrinkage of dendrites in mature pyramidal neurons. Further, disruption of Golgi polarity produces neurons with symmetric dendritic arbors lacking a single longest principal dendrite. These results define a novel polarized organization of neuronal secretory trafficking and demonstrate a mechanistic link between directed membrane trafficking and asymmetric dendrite growth.     

Significance of source and size in the mechanical response of human cerebral blood vessels

Cerebral blood vessels are frequently damaged in traumatic brain injury. Mechanical properties of fresh human cerebral vessels obtained through surgeries have been reported. Because surgical sources of human specimens are rare and produce a limited amount of material, we sought to compare the properties of more readily available cerebral arteries and veins obtained from cadavers to fresh vessel data. Additionally, because the previous study was limited to small vessels available in surgery, it was unknown how generally applicable the results were to larger cerebral arteries and veins. In the current study, large and small cerebral vessels from autopsy were stretched axially. Data from these and similar tests on fresh vessels were combined to determine the significance of source and size on mechanical properties. Structural comparisons of histological samples were additionally utilized to characterize differences. Results indicate that specimens from autopsy and surgery behave similarly except that vessels from autopsy tend to be less extensible. While tests on large vessels were limited, small arteries obtained from autopsy tended to be slightly stiffer than large arteries. In contrast, bridging veins from cadavers were typically stiffer and stretched less before structural failure than cortical veins from the same source. These effects are, however, secondary to differences identified between arteries and veins in the previous study.     

Growth hormone deficiency and replacement in patients with treated Cushing's Disease, prolactinomas and non-functioning pituitary adenomas: effects on body composition, glucose metabolism, lipid status and bone mineral density

BACKGROUND/AIMS: This study was designed to determine whether previous Cushing's disease (CD) or prolactinoma (PRL) could exert adverse effects additional to those of growth hormone (GH) deficiency as a consequence of variable degrees of prior hypogonadism or hypercatabolism. We report the effects of 5 years GH treatment in 124 GH deficiency adults; 42 patients with non-functioning pituitary adenomas (NFPA), 43 with treated PRL and 39 with treated CD. METHODS: Fasting plasma glucose, HbA(1c), lipoprotein profile, anthropometry and bone mineral density (BMD) were measured at baseline, 6 months and annually up to 5 years. RESULTS: Mean body mass index remained unchanged in the PRL group and tended to increase in the NFPA group. In contrast, body mass index decreased in the CD group. Decreases in waist and waist/hip ratio were seen in all groups at 6 months. Decreases in total cholesterol and low-density lipoprotein cholesterol were seen in all groups and remained sustained at 5 years. Plasma glucose and HbA(1c) increased at 6 months. Subsequently, plasma glucose returned to baseline values at 5 years; in contrast, HbA(1c )remained unchanged at the end of the study. Baseline lumbar spine and hip BMD were lower in the PRL and CD groups than in the NFPA group, decreased over 1 year in all groups and subsequently increased by 2 years in NFPA with a subsequent increase in lumbar spine BMD in PRL and CD groups delayed to 3-5 years. CONCLUSIONS: Baseline characteristics and response to GH replacement are qualitatively similar in NFPA, PRL and CD patients. Because improvements in BMD occur later in PRL and CD patients, an extended trial of GH therapy may be indicated in those patients who were commenced on GH therapy as an additional treatment for reduced BMD.     

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one (n = 31 recipients) or two (n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127=17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo (P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 microg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.     

A giant rhinocerotoid (Mammalia, Perissodactyla) from the Late Oligocene of north-central Anatolia (Turkey)

A giant rhinocerotoid is described for the first time south of the Black Sea, in Turkey. The single specimen, a fragmentary radius referred to Paraceratherium sp., originates from conglomerates nearby at Gözükizilli, in the Çankiri–Çorum Tertiary basin. These layers correspond to the Lower member of the Kizilirmak Formation. The same locality (Gözükizilli-2) yields also the small rhinocerotid Protaceratherium sp., cf. P. albigense (Roman, 1912). Three other mammal localities (Gözükizilli-1, in the Lower Member of the formation, with several rodent species; Tepe 641 and Kizilirmak, in the Upper Member, with a diversified micro- and macro-mammal fauna) allow us to refer the Kizilirmak Formation as a whole to the Late Oligocene. All the observed taxa have strong Asian and/or European affinities, which precludes any geographical insulation for this part of Anatolia during the Late Oligocene.  © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 152 , 581–592.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

Monitoring the positioning of short polycationic peptides in model lipid bilayers by combining hydrogen/deuterium exchange and electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to analyze the hydrogen/deuterium exchange properties of the mastoparan peptide Apoica-MP during interactions with lipid vesicle membranes. Synthetic peptide was incorporated into large unilamellar vesicles (LUVs) of L-alpha-phosphatidylcholine (PC), resulting in proteoliposomes which were then diluted with D2O. After quenching deuteration by the addition of formic acid the H/D exchange was directly analyzed by ESI-MS. This strategy was used to investigate the architecture of the peptide in the membranes of PC LUVs. The deuterated peptide ions were analyzed under collision-induced dissociation (CID) mass spectrometry, which permitted the location of deuterons at the amide sites along the peptide backbone. Intramolecular hydrogen scrambling was investigated both in the free peptide and in its proteoliposome form. Some scrambling was observed for the free peptide; however, almost no scrambling occurred in the amide hydrogens of the peptide backbone embedded in the membrane. The CID spectra suggest that the N-terminal moiety of the peptide lies on the polar side of the lipid membrane, while the C-terminal region is embedded in the membrane. The protocol described here may be reliably applied to investigate the interaction of mastoparans with bilayer lipid systems.     

Intracellular and extracellular labeling of neurons in fresh slice preparations combined with multiple tract tracing

We describe methods for studying axo-dendritic projections, one of the forms of neural connection involved in the complex circuits of the central nervous system, including brainstem auditory pathways. This form of neural connection is often difficult to visualize by conventional tract tracing techniques. Retrogradely identified cells were filled intracellularly with a mixture of fluorescent Lucifer yellow and nonfluorescent HRP in live slice preparations to reveal the detailed morphological features of these cells with special attention to the distal dendrite that may receive projections from suspected or known input axons. Extracellular or intracellular labeling of cells with axons that project to the distal dendrite of the identified cells was accomplished in the same live slice preparation. Using a live slice rather than a fixed slice allows accurate, visually controlled placement of anterograde tracer, which requires living axons for transport, into the source of input to the identified cells within the slice. Live slices also permit one to characterize the identified cells electrophysiologically. Intracellular labeling of cells in a potential source of local input to the identified cells also provides conclusive information concerning with connections of the cells involved.