Variant exons v6 and v7 together expand the repertoire of glycosaminoglycans bound by CD44

Isoforms of the glycoprotein CD44 are cell surface receptors for the glycosaminoglycan hyaluronate. They have been implicated in many biological processes, but their function in these is poorly understood and cannot be explained solely by hyaluronate binding. In the present work we examine the ligand binding properties of alternatively spliced CD44 variant isoforms which are functionally involved in the immune system, embryonic development, and tumor behavior. We show that these isoforms bind directly to the purified glycosaminoglycans chondroitin sulfate, heparin, and heparin sulfate, in addition to being able to bind to hyaluronate. Binding to this extended repertoire of glycosaminoglycans by CD44 depends on the inclusion of peptide sequences encoded by the alternatively spliced exons v6 and v7, and occurs both when the CD44 is solubilized from the plasma membrane and when it is expressed on intact cells. A single point mutation in the most N-terminal hyaluronate binding motif of CD44 ablates both hyaluronate and chondroitin sulfate binding, suggesting that glycosaminoglycans are bound through a common motif, and that only one of the hyaluronate binding motifs is responsible for the majority of glycosaminoglycan binding by CD44 on the cell surface. Taken together, these observations indicate that alternative splicing regulates the ligand binding specificity of CD44 and suggest that structural changes in the CD44 protein have a profound effect on the range of ligands to which this molecule can bind with potentially wide-ranging functional consequences.     

Dimethoxyphenylethylamine and tetrahydropapaverine are toxic to the nigrostriatal system

Experimental allergic neuritis (EAN) is a T cell mediated animal model of Guillain-Barré syndrome, characterized by inflammation and demyelination of the peripheral nervous system (PNS). To study the involvement of immunoregulatory cytokines, we induced EAN in Lewis rats by immunizing with bovine PNS myelin (BPM) and Freund's complete adjuvant. mRNA expression of the cytokines IL-1beta, IL-6, IL-10, IL-12, TNF-alpha and TNF-beta, and the cytolytic effector molecule cytolysin was examined in lymph node mononuclear cells (MNC) over the course of EAN by in situ hybridization after culture without antigen and in the presence of BPM, the myelin P2 protein, the control antigen acetylcholine receptor, or the mitogen PHA. Three patterns of cytokine mRNA expressing MNC in relation to clinical EAN could be distinguished: (i) IL-1beta mRNA expressing cells peaked already on day 3 post immunization (p.i.), and BPM- and P2-reactive TNF-alpha, and BPM-reactive IL-6 mRNA expressing cells were also detected already on day 7 p.i., i.e., before onset of clinical EAN; (ii) BPM- and P2-reactive TNF-alpha peaked together with P2-reactive TNF-beta, IL-6 and IL-12 mRNA expressing cells at height of clinical EAN, consistent with a disease-promoting role for these four cytokines; (iii) high levels of BPM- and P2-reactive IL-10 and cytolysin mRNA expressing cells were observed only during recovery (day 28 p.i.), consistent with a disease down-regulating role of IL-10 and cytolysin. The results suggest a major proinflammatory role for IL-1beta, TNF-alpha, TNF-beta, IL-6 and IL-12 and a disease down-regulating function of IL-10 as well as cytolysin in EAN.     

Role of neutrophil elastase in allergen-induced airway microvascular leakage in sensitized guinea pigs

To determine the role of neutrophil elastase in allergen-induced airway microvascular leakage, we assessed vascular permeability of guinea pig trachea by measuring the extravasation of Evans blue dye in the circulating blood. Inhalation of ovalbumin (OA) to guinea pigs sensitized with OA caused Evans blue extravasation, indicating an increased microvascular permeability. Pretreatment with ONO-5046 a specific inhibitor of neutrophil elastase, inhibited OA-induced vascular leakage in a dose-dependent manner. Tracheal instillation of human neutrophil elastase likewise increased microvascular permeability, and this effect was almost completely abolished by ONO-5046. Challenge with OA increased the number of neutrophils and neutrophil elastase activity in the bronchoalveolar lavage fluid, and these effects were inhibited by ONO-5046. These results suggest that neutrophil accumulation into the airway and the subsequent release of neutrophil elastase may play a role in the airway microvascular leakage produced by antigen challenge.     

Analysis of Vero toxins 1 and 2 by high-performance liquid chromatography/electrospray ionization mass spectrometry

PURPOSE: To study the interaction between gabapentin (GBP) and high-protein meals, 12 patients with epilepsy were administered this drug both while in a fasting state and after a high-protein meal. METHODS: After having acquired their informed consent, the patients (suffering from partial complex seizures resistant to other anticonvulsants) were randomly assigned to 2 groups of 6 subjects. Each subject was treated in a fasting state with a single 400 (group A) or 800 (group B) mg GBP oral dose. After 24 h, the GBP dose regimen was repeated, but was given after a high-protein meal. Serum GBP concentrations were measured by LC-Mass at baseline and 0.5, 1, 2, 3, 5, 7, 9, 12, and 24 h. Saliva GBP concentrations were determined at baseline and 2, 4.8, and 12 h. GBP urinary excretion was determined at 0-4, 4-8, and 8-12 h intervals. The following kinetic parameters were calculated: area under the concentration time curve from zero time to 24 h after the dose, AUC 0-24 h; maximal serum concentration, Cmax; time to the maximal serum concentration, Tmax; absorption rate constant, ka; elimination rate constant, beta; elimination half-time, t1/2beta. Student's t test for paired data, with significance assigned at P < 0.05, was used. RESULTS: No statistically significant differences were seen in GBP serum or saliva concentrations or in its urinary excretion (both in A or B group) between fasting and after the high-protein meal. CONCLUSIONS: High-protein meals do not seem to interfere with oral disposition of GBP.     

Tensile and impact properties changes of HASTELLOY X after exposure in high-temperature helium environment

Experimental results are presented on the changes in mechanical properties of HASTELLOY X* after being used in the liner tube of HENDEL hot gas duct under high temperature helium gas for about 6000 hours. In both room and elevated-temperature tensile tests, 0.2 pct proof stress and total elongation were significantly decreased after exposure in high temperature helium. Room-temperature impact toughness of the exposed specimens exhibited a much lower absorbed energy (about 5.0 × 10⁶ J/m²) than that of unexposed specimens (about 1.5 × 10⁶ J/m²). The fracture modes of tensile test specimens were more closely correlated with the test temperature than with the long-time exposure; however, long-time exposure is more affected by the tensile strength of HASTELLOY X than the test temperature. Moreover, “downstream effect” for the carbide precipitation reaction occurred in the liner tube of the HENDEL hot gas duct. HASTELLOY is a trademark of Cabot Corporation.     

Occlusal stability in Class II, Division 1, deep bite cases followed up for many years after orthodontic treatment

This case report analyzes long-term occlusal stability that can be achieved in Class II, Division 1, deep bite cases with active treatment finished during the period of maxillomandibular growth. The analysis was designed to identify occlusal features common to two cases at the end of active treatment and to study how the occlusion changed with growth and jaw movement to achieve stability. The following occlusal features were shared by the two cases at the end of active treatment: (1) AB plane and axes of the maxillary and mandibular posterior teeth were perpendicular to functional occlusal plane; (2) the axis of the lower incisor was almost perpendicular to DC-L1i line; (3) the anterior occlusion was overcorrected to or near an edge-to-edge relationship. Items 1 and 2 remained unchanged throughout the follow-up periods, regardless of growth status, and the overjet and overbite increased during maxillomandibular growth after treatment. During the period of mandibular growth alone, after the end of retention, the axes of maxillary incisors tipped labially; as a result, F line became parallel to CDM line by the end of growth. The labial tipping of maxillary incisors brought the lower incisal edge into contact with or extremely near the inflection point (Bp).1 By the end of growth, the tangent of Bp became parallel to or coincident with DC-L1i line and perpendicular to the axis of the lower incisor, and the DC-L1i lines at various times posttreatment were almost parallel to each other in the two cases. Overjet increased as the maxillary incisors tipped labially, providing proper protrusive and retrusive paths for mandibular guidance. The angle between the functional occlusal plane and CDM line stayed almost the same as at the end of active treatment in the two cases, suggesting a possible change in the angle of eminence in harmony with the functional occlusal plane. These factors apparently contributed to the long-term occlusal stability in the two cases.     

Expression of chemokine genes during rejection and long-term acceptance of cardiac allografts

Chemokines are cytokines with chemoattractant properties for leukocytes. They may play a critical role in directing leukocytes to graft sites and in amplifying intragraft inflammation during rejection. Previous studies have tested the intragraft expression of chemokine genes during the rejection of allogeneic skin grafts in mice. In the current study, we used a heterotopic heart transplant model in mice to test the intragraft expression of these genes in nonrejecting cardiac isografts, rejecting cardiac allografts, and cardiac allografts that were accepted due to immunosuppression with gallium nitrate. With the exception of low levels of interleukin-1beta and JE, intragraft expression of the the proinflammatory cytokine genes was not observed in either isografts or native heart. Two distinct patterns of chemokine mRNA were observed in the rejecting cardiac allografts. Intra-allograft expression of interleukin-1beta, interferon-gamma-inducible protein, JE, and KC was prominent by day 3 after transplantation. The expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated upon activation, normal T cell expressed and secreted (RANTES) was at low or undetectable levels at day 3 after transplantation but at high levels by day 8 after transplantation. Sixty days after transplantation, intra-allograft expression of chemokines in hearts from gallium nitrate-treated recipients indicated low levels of MIP-1alpha, MIP-1beta, and KC but high levels of interferon-gamma-inducible protein and RANTES.     

Increased activity of oleate-dependent type phospholipase D during actinomycin D-induced apoptosis in Jurkat T cells

Apoptosis is an active form of cell death that can be induced by a wide variety of agents and conditions. In response to actinomycin D, hydrogen peroxide (H2O2), or TNF-alpha, Jurkat T cells underwent typical apoptosis. Phospholipase D (PLD) activity in intact cells determined by phosphatidylbutanol generation was up-regulated by these agents. The PLD activation was in a time-dependent manner during apoptosis. It was also shown that the PLD activity measured by using exogenous substrate in the lysate from apoptotic cells was higher than that in the lysate from control untreated cells. The PLD activity in lysate from control untreated cells was stimulated by unsaturated fatty acids (UFA), but not by guanosine 5'-O-(3-thiotriphosphate). However, the PLD activity in the apoptotic cell lysate was no longer enhanced by the addition of oleate, suggesting that the increased PLD activity during apoptosis was attributed to the PLD of UFA-dependent type, but not the small G protein-dependent one. In fact, the release of free UFA was increased during apoptosis. The caspase inhibitors, z-DEVD and z-VAD, effectively suppressed PLD activation and apoptosis, but UFA release was unaffected. These results suggest the possibility that UFA-dependent type PLD may be implicated in apoptotic process in Jurkat T cells. This is the first demonstration that the PLD of UFA-dependent type would be involved in cellular responses.     

Multivesicular release at single functional synaptic sites in cerebellar stellate and basket cells

The purpose of the present work was to test the hypothesis that no more than one vesicle of transmitter can be liberated by an action potential at a single release site. Spontaneous and evoked IPSCs were recorded from interneurons in the molecular layer of cerebellar slices. Evoked IPSCs were obtained using either extracellular stimulation or paired recordings of presynaptic and postsynaptic neurons. Connections were identified as single-site synapses when evoked current amplitudes could be grouped into one peak that was well separated from the background noise. Peak amplitudes ranged from 30 to 298 pA. Reducing the release probability by lowering the external Ca2+ concentration or adding Cd2+ failed to reveal smaller quantal components. Some spontaneous IPSCs (1.4-2.4%) and IPSCs evoked at single-site synapses (2-6%) were followed within <5 msec by a secondary IPSC that could not be accounted for by random occurrence of background IPSCs. Nonlinear summation of closely timed events indicated that they involved activation of a common set of receptors and therefore that several vesicles could be released at the same release site by one action potential. An average receptor occupancy of 0.70 was calculated after single release events. At some single-site connections, two closely spaced amplitude peaks were resolved, presumably reflecting single and double vesicular release. Consistent with multivesicular release, kinetics of onset, decay, and latency were correlated to IPSC amplitude. We conclude that the one-site, one-vesicle hypothesis does not hold at interneuron-interneuron synapses.