Ligand Binding Characteristics of a Glycosylphosphatidyl Inositol Membrane-Anchored HeLa Cell Folate Receptor Epitope-Related to Human Milk Folate Binding Protein

The folate receptor (FR) in HeLa cells was characterized as to ligandbinding mechanism, antigenic properties and membrane anchor in order toobtain information to be used for the design of biological agentstargeting FR in malignant tumors. The receptor displayed the followingbinding characteristics in equilibrium dialysis experiments(37°C, pH 7.4) with [³H] folate: a high-affinity type of bindingthat exhibited positive cooperativity with a Hill coefficient >1.0and an upward convex Scatchard plot, a slow radioligand dissociation atpH 7.4 becoming rapid at pH 3.5 and inhibition in the presence of otherfolates. The molecular size of the receptor was 100 kDa on gel filtrationwith Triton X-100, or similar to that of high molecular weight human milkfolate binding protein (FBP). The latter protein represents a 25 kDamolecule which equipped with a hydrophobic glycosylphosphatidylinositol (GPI) membrane anchor susceptible to cleavage byphosphatidylinositol specific phospholipase C (PI-PLC) formsmicelles of 100 kDa size with Triton X-100. The HeLa cell FRimmunoreacted with antibodies against purified human milk FBP inELISA, and in a fluorescence activated cell sorting system, whereHeLa cells exposed to increasing concentrations of antibody showed adose-dependent response. Exposure to PI-PLC decreased the fraction ofimmunolabeled cells indicating a linkage of FR to cell membranes by aGPI anchor. HeLa cells incubated with radiofolate showed a continuousuptake with time, however, with a complete suppression of uptake in thepresence of an excess of cold folate. Prewash of cells at acidic pH toremove endogenous folate increased the uptake. Binding and uptake of [³H]folate was increased in cells grown in a folate-deprived medium. The HeLaFR seems to be epitope related to human milk FBP.     

Iodine speciation in human serum by reversed-phase liquid chromatography-ICP-mass spectrometry

Reversed-phase liquid chromatography-inductively coupled plasma mass spectrometric hyphenation was used for iodine speciation in human serum. First investigations showed that iodine species nearly quantitatively were eluted in the void volume. The result indicated that protein-linked thryoid hormones were not interacting with the stationary phase, thus being not retained. Investigations were performed about T4-TBG (thyroxin-thyroxin-binding globulin) complex generation and its retention during chromatography. It was shown that T4-TBG was not retained on the column. Therefore, a protease treatment was introduced for serum sample preparation. The analysis of “normal” sera (after protease) gave reasonable results lying in the range published in literature: I-:11; di-iodothyrosine (DIT): 2.1; mono-iodothyrosine (MIT): 1.6; reversed tri-iodothyronine (rT3): 3.9; T3: 5.9; T4: 60; each micrograms iodine per liter. The method also proved to recognize abnormalities in a pathologic serum, having rT3 as the predominant species. In this case the method obviously was superior compared to standard immunoassay methods, as it is monitoring the iodine in the species (physiologically active iodine species), whereas immunoassay methods may sometimes detect deiodinated (inactive) compounds.     

Guanylyl cyclase-C receptor mRNA distribution along the rat nephron

Guanylin (GN) and uroguanylin (UGN) are two recently identified peptides that have been shown to affect water and electrolyte transport in both the intestine and the kidney. Mechanistically, the effects of both peptides are thought to be mediated by intracellular cGMP which results from ligand binding to a plasma membrane guanylyl cyclase-C (GC-C) receptor. To date, the specific intrarenal site(s) of GN and UGN action have not been established. To begin to address this issue, the present studies utilized semi-quantitative RT-PCR to assess the distribution of GC-C mRNA in specific microdissected segments of the rat nephron. GC-C mRNA expression was highest in the cortical collecting tubule, followed by the proximal convoluted tubule, medullary thick ascending limb and collecting tubule, and thin limbs of Henle's loop. Expression levels were significantly lower in all other segments tested, including the glomerulus. The renal tubular expression pattern for cGMP-dependent protein kinase II (cGK-II) mRNA, which is activated in response to GN/UGN-dependent cGMP accumulation, was similar to that for GC-C. Notably, both GN and UGN mRNAs were also expressed along the nephron. The highest levels of expression for both peptides were detected in the medullary collecting tubule. Lower, but comparable levels of GN and UGN expression also occurred in the cortical collecting tubule, cortical and medullary thick ascending limb, and thin limbs of Henles loop. In the proximal convoluted tubule, GN mRNA expression was also quite high, while UGN mRNA was almost undetectable. The presence of renal GC-C and cGK-II in the kidney are consistent with a proposed endocrine function for GN and UGN. In addition however, the present data suggest that intrarenally synthesized GN and UGN may also contribute to the regulation of renal tubular transport.     

L-prolyl-l-leucyl-glycinamide and its peptidomimetic analog 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) attenuate haloperidol-induced c-fos expression in the striatum

Acute treatment of rats with haloperidol results in a rapid and transient increase in striatal c-fos mRNA and Fos immunoreactivity. The induction of immediate early genes by haloperidol may be involved in the development of extrapyramidal side effects. L-Prolyl-L-leucyl-glycinamide (PLG, or MIF-1) has been observed to antagonize the development of haloperidol-induced D(2) receptor supersensitivity in rats. We investigated the modulatory effects of PLG on haloperidol-induced c-fos and Fos protein expression in the rat striatum. We report that coadministration of either PLG or the potent analog of PLG, 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetam ide (PAOPA), attenuated haloperidol-induced c-fos and Fos expression. Haloperidol induced [2 mg/kg, intraperitoneally (i.p.)] c-fos and Fos expression by 500% and 100%, respectively. These responses were attenuated by 170% and 75%, respectively, when coadministered with PLG (20 mg/kg, i.p.) or by 79% by PAOPA (10 microg/kg, i.p.).     

Pollen limitation of reproduction in a subarctic-alpine population of Diapensia lapponica (Diapensiaceae)

Determinants of reproductive success (measured as capsule mass) in Diapensia lapponica , an early-flowering pollen-risker dependent on insects for pollination, were evaluated in an exposed subarctic-alpine population in northern Sweden. The species was protogynous and self-compatible, but self-deposition of pollen was inefficient. Hand-pollination increased capsule mass significantly, indicating that reproductive success was severely pollen limited. An experiment designed to test the relative importance of insect pollination in comparison to water availability (precipitation) and shelter (temperature and wind), revealed that exclosure of insects decreased capsule mass significantly, as did watering, whereas shelter tended to increase capsule mass. Pollination was the most important determinant of reproductive success, and the study suggests that pollen limitation may be particularly severe for early-flowering species, i.e. pollen-riskers, in exposed locations.     

Dolichol is the major lipid component of human substantia nigra neuromelanin

Neuromelanin is a dark brown pigment present at high concentrations in dopaminergic neurones of the human substantia nigra (SN). Early electron microscopic examinations of neuromelanin fine structure revealed a significant neutral lipid component; however, the identity of this lipid has remained unknown. Here we show that the lipid component of neuromelanin pigment derived from human SN is the polyisoprenoid dolichol. Established methods were used to isolate the pigment from the SN of 32 brains and the lipid fraction was recovered in high purity and yield. Using reversed-phase HPLC, atmospheric pressure chemical ionization mass spectrometry, and 1H- and 13C-NMR techniques, we showed that the neuromelanin dolichol contained 17-23 isoprenoid units. Dolichol accounted for 14% of the mass of neuromelanin pigment; low levels of other hydrophobic compounds were detected (e.g. ubiquinone-10, alpha-tocopherol and cholesterol together accounted for < 0.5% of the neuromelanin lipid mass). This is the first time that dolichol has been identified in such a physiological setting and significantly advances our understanding of neuromelanin pigment structure and biosynthetic pathways. Furthermore, these studies identify a potential novel role for the isoprenoid pathway in the regulation of neuromelanin function and neurodegeneration within the SN.     

Acute exercise increases the ventricular arrhythmia threshold via the intrinsic adenosine receptor system in conscious hypertensive rats

Coronary artery occlusion-induced tachyarrhythmias that culminate in ventricular fibrillation are the leading cause of death in developed countries. The intrinsic adenosine receptor system protects the heart from an ischemic insult. Thus the increased functional demands made on the heart during exercise may produce protective adaptations mediated by endogenous adenosine. Therefore, we tested the hypothesis that a single bout of dynamic exercise increases the ventricular arrhythmia threshold (VAT) induced by coronary artery occlusion in conscious hypertensive rats via the intrinsic adenosine receptor system. To test this hypothesis, we recorded the VAT before and on an alternate day after a single bout of dynamic treadmill exercise (12 m/min, 10% grade for 40 min). A single bout of dynamic exercise significantly reduced postexercise arterial pressure (Delta-24 +/- 4 mmHg) and increased VAT (Delta+1.95 +/- 0.31 min). Adenosine receptor blockade with the nonselective adenosine receptor antagonists theophylline or aminophylline (10 mg/kg) attenuated the cardioprotective effects of a single bout of dynamic exercise. Results suggest that strategies that increase myocardial ATP requirements leading to adenosine production provide protection against coronary artery occlusion.