Spider mechanoreceptors

Spiders have highly developed mechanosensory systems, some of which provide access to forms of stimulation alien to our own sensations. Studies of hair-shaped air movement detectors (trichobothria) and tactile sensors have uncovered an outstanding refinement of the processes of stimulus uptake and stimulus transformation, which reflect details of both stimulus physics and behavioral significance. They also emphasize the potential contained in the seemingly simple Bauplan of arthropod cuticular hairs. Embedded into the spider exoskeleton are several thousands of strain detectors (slit sensilla) measuring compressive exoskeletal strains induced by various forms of loads and forces. A compound slit sensillum (lyriform organ) on the leg has become an important model system for studies of mechanoreceptor primary processes at the cellular and membrane level.     

Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin

Cross-reactive carbohydrate determinants of plants are essentially a mixture of N-glycans containing beta1,2-xylose and core alpha1,3-fucose, the latter also found in insect glycoproteins. To determine the relative contributions of these two sugar residues to antibody binding, we prepared an array of glycomodified forms of human apo-transferrin. Using core-alpha1, 3-fucosyltransferase (EC 2.4.1.214) and beta1,2-xylosyltransferase (EC 2.4.2.38) recombinantly expressed in Pichia pastoris and suitable glycosidases, glycoforms containing either only fucose (MMF), only xylose (MMX), both (MMXF), or neither (MM) linked to the common pentasaccharide core were generated. Additional glycoforms were obtained by enzymatic removal of the alpha1,3-linked mannosyl residue. These transferrin glycoforms served to define the binding specificity of antibodies in western blot, ELISA, and inhibition ELISA. Rabbit anti-horseradish peroxidase serum bound to both the fucosylated (MMF) and the xylosylated (MMX) glycoforms. Inhibition studies indicated two independent highly specific populations reacting with either of the two epitopes. In contrast, the monoclonal antibody YZ1/2.23 appears to recognize a larger structure including both the fucosyl and the xylosyl residue. The mannose-deficient glycoform was a poorer inhibitor for both antibodies. Terminal GlcNAc residues prevented antibody binding. Rabbit anti-bee venom serum reacted with fucosylated forms (MMF and MMXF) only. Experiments with sera from allergic patients suggest that glycomodified human transferrin, especially the MMXF glycoform, is a suitable reagent for the detection of antibodies against cross-reactive carbohydrate determinants. Within the panel studied, several sera contained high levels of fucose-reactive IgE but only a few sera showed any binding to MMX-transferrin.     

Phenotypical evidence for a gender difference in cardiac norepinephrine transporter function

Norepinephrine transporter (NET) function has a central role in the regulation of synaptic norepinephrine concentrations. Clinical observations in orthostatic intolerance patients suggest a gender difference in NET function. We compared the cardiovascular response to selective NET inhibition with reboxetine between 12 healthy men and 12 age-matched women. Finger blood pressure, brachial blood pressure, and heart rate were measured. The subjects underwent cardiovascular autonomic reflex testing and a graded head-up tilt test. In a separate study, we applied incremental concentrations of tyramine and isoproterenol through subcutaneous microdialysis catheters in eight men and in eight women. NET inhibition elicited a threefold greater increase in supine blood pressure in men than women (P < 0.05). The pressor response was driven by an increased cardiac output. The orthostatic heart rate increase during NET inhibition was greater in men than women (56 +/- 5 beats/min in men, 42 +/- 4 beats/min in women, P < 0.001). In contrast, NET inhibition resulted in a similar suppression in the cold pressor and handgrip response, low-frequency blood pressure oscillations, and venous norepinephrine in the supine position. Men and women were similarly sensitive to the lipolytic effect of isoproterenol and tyramine. We conclude that NET inhibition results in more pronounced changes in cardiac regulation in men than women. Our observations suggest that the NET contribution to cardiac norepinephrine turnover may be decreased in women. The gender difference in NET function may not be expressed in tissues that are less NET dependent than the heart.     

Immunocytochemical localization of Na+-HCO3- cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells

In corneal endothelium, there is evidence for basolateral entry of HCO(3)(-) into corneal endothelial cells via Na(+)-HCO(3)(-) cotransporter (NBC) proteins and for net HCO(3)(-) flux from the basolateral to the apical side. However, how HCO(3)(-) exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO(3)(-) similarly to fresh tissue. In addition, Cl(-) channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl(-) channels, CO(2) diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.     

Mechanisms of ADRF release from rat aortic adventitial adipose tissue

Blood vessels are surrounded by variable amounts of adipose tissue. We showed earlier that adventitial adipose tissue inhibits rat aortic contraction by release of a transferable factor, adventitium-derived relaxing factor (ADRF), which activates smooth muscle K(+) channels. However, little is known about the mechanisms of ADRF release. Using isolated rat aortic rings and isometric contraction measurements, we show that ADRF release depends on extracellular [Ca(2+)] (EC(50) approximately 4.7 mM). ADRF effects do not involve neuronal presynaptic N-type Ca(2+) and Na(+) channels or vanilloid, cannabinoid, and CGRP receptors. ADRF release is strongly inhibited by the protein tyrosine kinase inhibitors genistein and tyrphostin A25. In contrast, daidzein, an inactive genistein analog, and the protein tyrosine kinase inhibitor ST638 had no effect. Protein kinase A inhibition by H89 also inhibited ADRF release, whereas the protein kinase G inhibitor KT-5823 had no effect. We propose that ADRF release is Ca(2+) dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. Furthermore, ADRF release does not depend on perivascular nerve endings.     

Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.     

Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Determination of dihydroergocryptine in human plasma and urine samples using on-line sample extraction-column-switching reversed-phase liquid chromatography-mass spectrometry

A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.     

Carbohydrate moieties can induce mediator release: a detailed characterization of two major timothy grass pollen allergens

Specific IgE binding to carbohydrate moieties of glycosylated allergens has been known for years, but the importance of these structures for the elicitation of allergic reactions is still a matter of debate. Because of their conserved carbohydrate structures, especially N-glycans have always been prime candidates for IgE cross-reactivity between allergens from unrelated species. The aim of our study was to determine whether carbohydrate structures on glycoproteins can by themselves elucidate allergic reactions. We characterized in detail the carbohydrate moieties of the major allergens Phl p 1 and Phl p 13 of timothy grass pollen (Phleum pratense L.) by performing tryptic digests followed by HPLC, N-terminal sequencing, sugar analysis, MALDI-TOF- and ESI-ICRFT-MS. Phl p 1 contains one N-glycan with one of the two glycoforms MMXF3 and M0XF3 and a single furanosidic arabinose, which is bound to a hydroxyproline residue in direct vicinity to the N-glycan. This O-glycosylation is probably due to an arabinosylation consensus sequence found in the N-terminal part of Phl p 1 and other group 1 allergens, but displayed no IgE-reactivity. Thus, Phl p 1 is monovalent with respect to its IgE-binding carbohydrate epitopes and showed no mediator release. In contrast, the carbohydrate moiety of Phl p 13, which carries four of the same N-glycans (like Phl p 1), can cross-link IgE-receptors via carbohydrate chains and elicits IL-4 release from basophils.     

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.