Further characterization of malignant glioma-derived vascular permeability factor

The nature of vascular permeability factor (VPF) activity derived from serum-free conditioned medium containing cultured human malignant glial tumors has been further investigated. A 1000-fold purification was accomplished by sequential heparin-Sepharose affinity chromatography and high-performance liquid chromatography gel filtration chromatography steps. Vascular permeability factor activity falls into a molecular weight range of 41,000 to 56,000 D. Activity is bound to hydroxylapatite, carboxymethyl-Sepharose, phenyl-Sepharose, and heparin-Sepharose, whereas little or no activity was bound to diethylaminoethyl-Sephacel. Vascular permeability factor activity is trypsin- and pepsin-sensitive but is unaffected by treatment with ribonuclease A. This suggests that VPF is a hydrophobic, positively charged (cationic) polypeptide with a potentially biologically significant affinity for heparin. As most proteins are negatively charged (anionic) and have no affinity for heparin, a significant advantage was gained by performing these purification steps. The activity of VPF is not inhibited by coinjection of conditioned medium with soybean trypsin inhibitor; or hexadimethrine (both known antagonists of tissue plasminogen activator, Hageman factor, and serum kallikrein); or aprotinin (an antagonist of both plasmin and tissue kallikrein); or phenylmethanesulfonyl fluoride (a serine esterase (elastase) inhibitor); or pepstatin-A (an acid protease inhibitor which inactivates vascular permeability-inducing leukokinins). These data, together with the fact that VPF is produced and released into serum-free media, provides substantial evidence against it being one of the more commonly known serum-derived permeability mediators. Treatment with dithiothreitol inhibited VPF activity, indicating the presence of at least one essential disulfide bond in this molecule. Inhibition by dexamethasone of VPF expression in cultured malignant glial cells appears to be selective. Dexamethasone-induced inhibition of VPF was dose-responsive and was not associated with a parallel inhibition of cellular protein synthesis as determined by tritiated leucine incorporation into trichloroacetic acid-precipitable material. Inclusion of dexamethasone in the culture medium was not associated with altered cell viability or cell number. A series of in vivo studies confirmed the inhibition of VPF activity in test animals pretreated with dexamethasone. This steroid-induced inhibition was partially reversed by treatment of test animals with actinomycin D prior to exposure to dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhancing health-care research: an interdisciplinary collaborative approach.

Many research programs tackle complex problems that cannot be comprehensively investigated by a sole researcher or a research team from a single profession. Interdisciplinary teams can develop a collective mass of common knowledge, broaden the scope of research, and produce more clinically relevant outcomes that are sensitive to the realities of practice. The authors describe the experience of a research team from the perspective of its members. The purposes of the paper are to highlight the benefits of an interdisciplinary collaborative approach to research and to describe the characteristics of a successful team. Some of the benefits discussed include increased research productivity and quality, professional development and mentorship, support and encouragement, expanded resource networks, and bridging of the gap between academia and practice. The authors also discuss the characteristics of a successful research team, associated challenges, and recommendations for enhancing research endeavours through collaboration.     

差示—线性组合导数光谱法测定扑咳灵片中扑尔敏和溴化丙胺太林

本文提出了差示一线性组合导数光谱法用于测定混合组分体系的基本原理和实验方法。并试用本法消除了扑尔敏、溴化丙胺太林、灵芝粉及附加剂之间的相互干扰,从而分别测定了扑咳灵片巾扑尔敏和溴化丙胺太林的含量,其平均回收率分别为100.2±1.49％(CV)和99.89±1.03％(CV)。其精密度、准确度均好。     

Cytosolic calcium changes in endothelial cells induced by a protein product of human gliomas containing vascular permeability factor activity

A vascular permeability factor (VPF) derived from serum-free conditioned medium of cultured human malignant gliomas (HG-VPF) has been described previously. The rapid kinetics of HG-VPF activity in an in vivo assay of vascular permeability suggests a direct action upon the vascular endothelial cell. To determine whether HG-VPF was capable of inducing a physiologically significant alteration in isolated endothelial cells, cytosolic calcium [Ca++]i was measured in vitro in these cells before and after their exposure to media containing this substance. This was accomplished by preloading cultured endothelial cells with a fluorescent intracellular Ca++ probe fura-2/AM. It was found that HG-VPF induced a rapid and transient elevation of [Ca++]i in normal endothelial cells derived from human umbilical vein, bovine adrenal medulla, bovine pulmonary artery, and rat brain. This effect was inhibited by chelating extracellular calcium [Ca++]e with ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetra-acetic acid (EGTA), indicating that the HG-VPF-induced response resulted from the influx of extracellular calcium. The addition of cations that act as nonspecific calcium channel blockers (Li+, Co++, Mn++, La ) completely inhibited VPF activity, further supporting the role of [Ca++]e influx. The HG-VPF activity was not, however, blocked by verapamil, a calcium antagonist that appears to be specific for voltage-gated calcium channels. Furthermore, exposure of endothelial cells to 120 mM [K+]e did not result in a calcium transient. Coincubation of endothelial cells with dexamethasone inhibited HG-VPF-induced rises in [Ca++]i, while having no effect upon cyclic nucleotide-induced changes in calcium. The present studies indicate that vascular extravasation induced by human glioma-derived VPF may be mediated by a direct action upon vascular endothelial cells. Furthermore, the observed dexamethasone-induced inhibition of this process suggests a specific cellular action for corticosteroids. This, together with previous observations that dexamethasone suppresses both the production of VPF by tumor cells in vitro and its permeability-inducing activity in vivo, may explain the efficacy of glucocorticoids in the treatment of neoplastic vasogenic brain edema. Finally, studies with a polycationic peptide (protamine) known to induce blood-brain barrier disruption in vivo revealed similar effects upon endothelial cytosolic calcium levels. As HG-VPF is a positively charged macromolecule, a common interaction between these substances and the negatively charged endothelial cell surface in the induction of permeability is suggested. Nonspecific cross-linking of charged groups of the endothelial glycocalyx and specific HG-VPF receptor binding are both valid mechanisms of HG-VPF-mediated calcium changes. Their potential relevance in the setting of microvascular permeability is discussed.     

Penetration of recombinant interleukin-2 across the blood-cerebrospinal fluid barrier

Recombinant interleukin-2 (rIL-2) is an immunotherapeutic agent with efficacy against certain advanced cancers. The penetration of rIL-2 across the blood-cerebrospinal fluid (CSF) barrier was studied in 12 cancer patients who had no evidence of tumor involvement of the central nervous system. At different times during treatment with intravenous rIL-2, CSF was withdrawn either continuously for 8 to 26 hours via a lumbar subarachnoid catheter (in eight patients) or by a single lumbar puncture (in four). Bioassay showed the appearance of rIL-2 in lumbar CSF 4 to 6 hours after the first intravenous dose, a rise over 2 to 4 hours to a plateau of 3 to 9 U/ml, and clearance to less than 0.1 U/ml by 10 hours after the last dose. An abnormally elevated CSF albumin level in two of the twelve patients indicated alteration of the blood-brain barrier. There were no abnormalities in the CSF glucose level or white blood cell count. The CSF pharmacokinetics contrast with the rapid elimination of rIL-2 from plasma and demonstrate significant blood-CSF barrier penetration. These data support the possibility of achieving CSF levels of rIL-2 that are adequate to maintain activity of lymphokine-activated killer cells after parenteral administration, and argue for rIL-2-associated disruption of the human blood-brain barrier in some patients.     

Some observations on the catecholaminergic innervation of the intermediate zone of the thoracolumbar spinal cord of the cat

The anatomical arrangement of catecholaminergic nerve terminals in the intermediate zone of the thoracolumbar spinal cord was examined with the fluorescence microscope in serial sections of spinal cord from adult cats perfused with a formaldehyde-glutaraldehyde mixture. Specific fluorescence in this material was assumed to represent noradrenaline. The distribution of fluorescent varicose nerve terminals was compared with that of neuron cell bodies in the same sections after Nissl counterstaining, and with the known topographic distribution of sympathetic preganglionic neurons. Dense accumulations of noradrenergic fibers were found in the intermediate zone in all segments between the caudal part of C8 and the rostral part of L4. These were not only associated with preganglionic neurons in the intermediolateral columns (ILN), but below T5 also extended in bands (1-3 per mmm of spinal cord) toward, and in some segments across, the midline. Noradrenergic terminals were associated with cell bodies in most parts of the ILN from T1 to T8 and in L2-3. Between T9 and L2, the innervation of the ILN was patchy, and the majority of the noradrenergic fibers projected to regions medial to the ILN. These corresponded to sites at which preganglionic neurons and also possible interneurons of the intermediomedial nucleus occur. Although preganglionic neurons and clusters of noradrenergic terminals are located in similar regions across the intermediate zone, their densities and patterns of distribution differ. This observation applies to comparisons both between anatomical subnuclei and between segments in a characteristic way along the length of the thoracolumbar cord.     

Water intake and the neural correlates of the consciousness of thirst

Thirst and resultant water drinking can arise in response to deficits in both the intracellular and extracellular fluid compartments. Inhibitory influences mediating the satiation of thirst also are necessary to prevent overhydration. The brain regions that underpin the generation or inhibition of thirst in these circumstances can be categorized as sensory, integrative, or cortical effector sites. The anterior cingulate cortex and insula are activated in thirsty human beings as shown by functional brain-imaging techniques. It is postulated that these sites may be cortical effector regions for thirst. A major sensory site for generating thirst is the lamina terminalis in the forebrain. Osmoreceptors within the organum vasculosum of the lamina terminalis and subfornical organ detect systemic hypertonicity. The subfornical organ mediates the dipsogenic actions of circulating angiotensin II and relaxin. Major integrative sites are the nucleus of the tractus solitarius, the lateral parabrachial nucleus, the midbrain raphé nuclei, the median preoptic nucleus, and the septum. Despite these advances, most of the neural pathways and neurochemical mechanisms subserving the genesis of thirst remain to be elucidated.