Olfactory function in patients with hypogonadotropic hypogonadism: an all-or-none phenomenon?

Hypogonadotropic hypogonadism (HH) refers to an endocrine defectof hypothalamic origin resulting in gonadal hypoplasia and frequentlyassociated with anosmia or severely impaired olfactory function(Kallmann's syndrome). This apparently results from a disruptionin the migration of neurons from the olfactory placode to thebulb and hypothalamus early in development, and so providesa unique opportunity to investigate olfactory function in humansubjects with congenitally incomplete peripheral systems. Olfactoryperformance in 37 HH patients and 37 age-matched controls wascompared using a modified version of the Munich Olfaction Test.This test is based on the sniff-bottle method and includes testsof (i) odor quality discrimination, (ii) intensity discrimination,(iii) detection thresholds, and (iv) recognition, hedonic evaluationand identification ability. The patients could be divided intotwo distinct groups differing significantly on all four subtestsand showing no overlap in performance: 20 anosmics, conformingto Kallmann's syndrome, and 17 apparent normosmics whose performancewas slightly poorer, but not significantly different to thatof the controls. The unexpected failure to find a continuumof olfactory dysfunction now raises the question whether HHwith or without anosmia represents two syndromes with distinctetiologies, or rather reflects the ability of the olfactorysystem to function well despite morphological impairment.     

Specific binding sites for vasoactive intestinal polypeptide on nonadherent peripheral blood lymphocytes

Vasoactive intestinal polypeptide (VIP), an octacosapeptide isolated from porcine duodenum and thought to have neuromodulator function in several functional systems (gastrointestinal tract, brain, lung, genital tract, heart), was recently detected in human neutrophils by radioimmunoassay. Subsequent studies demonstrated a VIP-mediated increase in lymphocyte adenylate cyclase. In this paper, VIP binding studies are presented using viable nonadherent human lymphocytes. Binding of 125I-VIP to nylon wool column-purified lymphocytes is specific, time dependent, rapid, and reversible. Bound radioactivity varies linearly with the number of cells used and is displaceable by non-iodinated VIP in a dose-dependent manner with complete displacement between 1 pM and 50 nM. Scatchard analysis of competition experiments demonstrates one class of specific binding sites with a KD of 0.47 +/- 0.23 nM and a Bmax of 24.9 +/- 7.0 pM. This Bmax represents 1700 binding sites/cell. secretin, gastric inhibitory polypeptide, and glucagon did not effectively compete with 125I-VIP for binding sites. This is the first demonstration of VIP receptors in a purified population of human lymphocytes; the data suggest that VIP may modulate lymphocyte function.     

Alterations of Red Cell Membrane Properties in Nneuroacanthocytosis

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an “acanthocytic state” of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.     

Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis. The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.     

Extracellular signal-regulated kinase 1 interacts with and phosphorylates CdGAP at an important regulatory site

Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.