Behavioral development in okapis and giraffes

Observations on the behavioral development of two okapi calves and one giraffe calf were made at Brookfield Zoo. The following behaviors were monitored for 4 to 6 mo after birth; nursing duration and nursing attempts, mother-infant distance, bunting the mother's udder, lying, moving, maternal grooming, mother and infant autogrooming, object licking, tail chewing, and contact by others in the herd. Behaviors in both species showed oscillating patterns with high levels of mother-infant contact behaviors at 3–4 wk, 9–11 wk, and 14–15 wk in okapis. Giraffe infants showed similar oscillations with high periods of contact about 2–5 wk later than those in okapis. Other behaviors oscillated in concert with these, with specific correlations occurring between nursing behaviors and grooming behaviors. A main difference between okapi and giraffe development centered around maternal motivation during the high contact (regressive) periods. In okapis, after 10–12 wk there was a low rate of nursing success, whereas in giraffes the percentage of success in nursing rose with later behavioral oscillations. The regressive periods became conflict periods in okapis, whereas in the giraffe, the mother initiated the periods. This difference was in accordance with the unique strategy of infant rearing in wild giraffes in which there is an extended “hider” period when older calves are left together in shaded areas with an adult sentry. Field studies also indicated probable oscillations of mother-infant contact and a prolonged period of the mother initiating contact with her calf.     

Tandem repeats of the tetramer 5'-CAAT-3'present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemo philus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neis seria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)ⁿ encoded by multiple tandem repeats of 5′-CAAT-3′. This observation suggested that (SINQ)ⁿ might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5′-CAAT-3’repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)ⁿ is not a prerequisite for the enzymatic function of Lic2A and that the 5′-CAAT-3’repeats in lic2A function solely as a mechan ism for generating phase variation. This observation suggested that wide variation in the number of 5’-CAAT-3’repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5′-CAAT-3’repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)ⁿ indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.     

Regulation of insulin-like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3–20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 μM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or, alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI. © 1994 Wiley-Liss, Inc.     

Studies on the complex formed between glucagon and dicaprylphosphatidylcholine

The interaction between glucagon and dicaprylphosphatidylcholine (DCPC) was studied by fluorescence, circular dichroism and calorimetry, as well as by ¹H- and ³¹P-nuclear magnetic resonance. The water-soluble lipid-protein complex was also characterized by gel filtration and ultracentrifugation. The complex appeared to be monodisperse by sedimentation equilibrium measurements, with a molecular weight of (4.55 ± 0.57)·10⁴. This complex contained approximately 7 molecules of glucagon and 35 molecules of phospholipid. Proton-decoupled ³¹P-NMR spectra of the phospholipid in the lipid-protein complex display narrower resonances than those of sonicated vesicles of DCPC, and ¹H-³¹P coupling could be detected in proton coupled spectra. These NMR results, together with gel-filtration results, suggest that glucagon ‘solubilizes’ phospholipid aggregates, forming a lipid-protein complex which is smaller than sonicated preparations of DCPC. ¹H-NMR resonance of both the methionine methyl group (met-27) and the aromatic envelope of glucagon are broadened by the phospolipid, indicating that the C-terminal region and the aromatic residues are involved in the interaction with the phospholipid. Nuclear magnetic resonance titrations of the imidazole ring C(2) and C(4) protons of the histidine residue of glucagon show that DCPC lowers the pK of the imidazole. The alterations caused by the phospholipid in the far and near ultraviolet CD spectra of glucagon reflect, respectively, the increased helix content of the hormone and the fact that the aromatic residues are located in a more structured environment. The phospholipid also alters the fluorescence properties of glucagon, shifting the fluorescence emission maximum of the hormone to shorter wavelength, and enhancing its relative intensity. This suggests that the fluorophore is experiencing a more hydrophobic environment in the presence of the lipid. Binding of glucagon to the phospholipid was analysed by Scatchard plots of the enhancement of fluorescence caused by the phospholipid and showed that the equilibrium binding constants of glucagon to DCPC are (4.4 ± 0.5)·10⁴M^?1 and (7.5±0.5)·10⁴M^?1, at 15°C and 25°C, respectively. The average number of moles of phospholipid bound per mole of glucagon is 4.4±0.6. The isothermal enthalpy of reaction of glucagon with DCPC is ?20.5 kcal/mol of glucagon at 25°C and ?32.5 kcal/mol of glucagon at 15°C. The observed enthalpies can arise from glucagon-induced cyrstallization of the phospholipid, from the non-covalent interactions between the peptide and lipid as well as from the lipid-induced conformational change in the protein. These results demonstrate that, unlike the complexes formed between glucagon and phospholipids which form more stable bilayers, the complex formed between glucagon and DCPC is stable over a wide range of temperatures, including temperatures well above the phase transition.     

Semiquantitative immunoblots of membrane protein-epidermal growth factor

Membrane proteins or cytokines are sometimes difficult to isolate and purify. Our group recently concentrated on epidermal growth factor (EGF) protein expression studies. Mature EGF was initially identified from mouse submaxillary gland extract as a stimulator of eyelid opening and incisor eruption when injected into newborn mice and rats. The EGF precursor is a transmembrane protein with eight additional EGF-like repeats. Our previous study has shown that the EGF precursor without these eight EGF-like repeats (hEGF) was biologically active. Here, we introduce a modified method for rapid detection of hEGF. The membranous protein was directly extracted from various organs of transgenic mice (including the submandibular gland, kidney, liver, heart, and testis) with two different buffers and easily detected by semiquantitative immunoblotting.     

Integrin α2β1 Mediates Tyrosine Phosphorylation of Vascular Endothelial Cadherin Induced by Invasive Breast Cancer Cells

The molecular mechanisms that regulate the endothelial response during transendothelial migration (TEM) of invasive cancer cells remain elusive. Tyrosine phosphorylation of vascular endothelial cadherin (VE-cad) has been implicated in the disruption of endothelial cell adherens junctions and in the diapedesis of metastatic cancer cells. We sought to determine the signaling mechanisms underlying the disruption of endothelial adherens junctions after the attachment of invasive breast cancer cells. Attachment of invasive breast cancer cells (MDA-MB-231) to human umbilical vein endothelial cells induced tyrosine phosphorylation of VE-cad, dissociation of β-catenin from VE-cad, and retraction of endothelial cells. Breast cancer cell-induced tyrosine phosphorylation of VE-cad was mediated by activation of the H-Ras/Raf/MEK/ERK signaling cascade and depended on the phosphorylation of endothelial myosin light chain (MLC). The inhibition of H-Ras or MLC in endothelial cells inhibited TEM of MDA-MB-231 cells. VE-cad tyrosine phosphorylation in endothelial cells induced by the attachment of MDA-MB-231 cells was mediated by MDA-MB-231 α₂β₁ integrin. Compared with highly invasive MDA-MB-231 breast cancer cells, weakly invasive MCF-7 breast cancer cells expressed lower levels of α₂β₁ integrin. TEM of MCF-7 as well as induction of VE-cad tyrosine phosphorylation and dissociation of β-catenin from the VE-cad complex by MCF-7 cells were lower than in MDA-MB-231 cells. These processes were restored when MCF-7 cells were treated with β₁-activating antibody. Moreover, the response of endothelial cells to the attachment of prostatic (PC-3) and ovarian (SKOV3) invasive cancer cells resembled the response to MDA-MB-231 cells. Our study showed that the MDA-MB-231 cell-induced disruption of endothelial adherens junction integrity is triggered by MDA-MB-231 cell α₂β₁ integrin and is mediated by H-Ras/MLC-induced tyrosine phosphorylation of VE-cad.     

Effects of hydrological forcing on the structure of a tropical estuarine food web

River flow can impact which sources of particulate organic matter (POM) fuel estuarine food webs. Here, we used stable carbon (C) and nitrogen (N) isotope analyses to compare how contributions of different POM sources (terrestrial, estuarine, and marine) to the diets of zooplankton and juvenile fishes differed between low and high river flow conditions, as well as spatially across a tropical estuary, Hilo Bay, Hawaii, USA. Diets of zooplankton and juvenile fishes were affected by river flow conditions, but the magnitude and the change in the basal resources depended on the location of the station in the estuary relative to the ocean and the river mouths. Consumers from the station most isolated from the ocean and with groundwater and overland flow inputs, utilized a combination of estuarine and terrestrial POM during both low and high river flow conditions and exhibited less variability in their basal resources than stations with direct ocean exchange. Consumers from stations in the Bay most affected by ocean exchange and river inputs utilized a combination of estuarine, terrestrial, and marine POM during low flow conditions, but shifted to marine and terrestrial POM during high river flow conditions. This shift to using terrestrial POM during high river flow conditions was substantial and up to 40% higher than values measured in other estuaries. Factors suspected to be affecting which POM source(s) consumers use in Hilo Bay are gross primary production, biological availability of exported terrestrial OM, and estuarine bacteria biomass, all of which are affected by river flow. Overall, our results suggest that Hilo Bay's food web and possibly those from other tropical estuaries are vulnerable to changes in hydrology, which may be further enhanced by global climate change.     

Comparing transect survey and WSR‐88D radar methods for monitoring daily changes in stopover migrant communities

ABSTRACT For decades, researchers have successfully used ground‐based surveys to understand localized spatial and temporal patterns in stopover habitat use by migratory birds. Recent technological advances with WSR‐88D radar now allow such investigations on much broader spatial scales. Both methods are assumed to accurately quantify patterns in migrant bird communities, yet information is lacking regarding relationships between radar estimates of migration and different ground‐based monitoring methods. From 2005 to 2007, we monitored migrant communities on or near two Department of Defense installations in the spring (Ft. Polk Military Complex, LA; U.S. Army Test and Evaluation Command, Yuma Proving Ground, AZ) and on two installations in the fall (Ft. Polk Military Complex, LA; Eglin Air Force Base, FL) using both ground‐based transect surveys and radar imagery of birds aloft. We modeled daily changes in migrant abundance and positive and negative species turnover measured on the ground as a function of radar estimates of migrant exodus and input densities. Radar data were not significant predictors of any response variable in any season either in the southeastern or southwestern United States, indicating a disparity between the results obtained using different methods. Multiple unique sources of error associated with each technique likely contributed to the conflicting outcomes, and researchers should take great care when selecting monitoring methods appropriate to address research questions, effects of management practices, or when comparing the results of migration studies using different survey techniques.     

Estimating abundance of mountain lions from unstructured spatial sampling

Mountain lions (Puma concolor) are often difficult to monitor because of their low capture probabilities, extensive movements, and large territories. Methods for estimating the abundance of this species are needed to assess population status, determine harvest levels, evaluate the impacts of management actions on populations, and derive conservation and management strategies. Traditional mark–recapture methods do not explicitly account for differences in individual capture probabilities due to the spatial distribution of individuals in relation to survey effort (or trap locations). However, recent advances in the analysis of capture–recapture data have produced methods estimating abundance and density of animals from spatially explicit capture–recapture data that account for heterogeneity in capture probabilities due to the spatial organization of individuals and traps. We adapt recently developed spatial capture–recapture models to estimate density and abundance of mountain lions in western Montana. Volunteers and state agency personnel collected mountain lion DNA samples in portions of the Blackfoot drainage (7,908 km²) in west-central Montana using 2 methods: snow back-tracking mountain lion tracks to collect hair samples and biopsy darting treed mountain lions to obtain tissue samples. Overall, we recorded 72 individual capture events, including captures both with and without tissue sample collection and hair samples resulting in the identification of 50 individual mountain lions (30 females, 19 males, and 1 unknown sex individual). We estimated lion densities from 8 models containing effects of distance, sex, and survey effort on detection probability. Our population density estimates ranged from a minimum of 3.7 mountain lions/100 km² (95% CI 2.3–5.7) under the distance only model (including only an effect of distance on detection probability) to 6.7 (95% CI 3.1–11.0) under the full model (including effects of distance, sex, survey effort, and distance × sex on detection probability). These numbers translate to a total estimate of 293 mountain lions (95% CI 182–451) to 529 (95% CI 245–870) within the Blackfoot drainage. Results from the distance model are similar to previous estimates of 3.6 mountain lions/100 km² for the study area; however, results from all other models indicated greater numbers of mountain lions. Our results indicate that unstructured spatial sampling combined with spatial capture–recapture analysis can be an effective method for estimating large carnivore densities. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.