Calcitonin gene-related peptide increases blood flow and potentiates plasma protein extravasation in the rat knee joint.

1. The effects of calcitonin gene-related peptide (CGRP) and other vasoactive mediators of inflammation on blood flow in the synovial vessels and plasma protein extravasation into the knee (femoro-tibial) joint of the pentobarbitone-anaesthetized rat were measured. 2. Changes in synovial blood flow were estimated by 133xenon clearance from the synovial cavity. CGRP (0.1 pmol and 10 pmol) and prostaglandin E1 (PGE1; 3 pmol and 300 pmol) significantly increased clearance from the knee joint measured 5 min after intra-articular injection. Substance P (10 pmol) had no effect on synovial blood flow. 3. Intra-articular perfusion of the rat knee with CGRP at concentrations up to 0.1 mM, or PGE1 at concentrations up to 10 microM, did not increase plasma extravasation into the synovial cavity measured by accumulation of intravenously injected 125I-albumin in the perfusate. 4. Plasma extravasation into the knee was significantly increased by infusion of bradykinin (0.1 microM), 5-hydroxytryptamine (1 microM) and histamine (0.1 mM), compared with the contralateral joints in the same animals which were perfused with Tyrode solution. 5. Perfusion of the knee joint with substance P did not specifically induce 125I-labelled albumin accumulation in the synovial cavity even at doses that had systemic effects as observed by marked plasma extravasation into other tissues. 6. The increase in plasma extravasation induced by histamine (0.1 mM) was potentiated by co-infusion with CGRP (0.1 microM) and PGE1 (3 microM). However the response to a submaximal dose (0.1 microM) of bradykinin, which induced similar plasma extravasation to histamine (0.1 mM), was not increased by co-infusion with CGRP or PGE1.7. These results show that CGRP is a potent vasodilator in the rat knee. CGRP released from sensory nerves may act synergistically with mediators of increased vascular permeability to modify the inflammatory response in this site.


Long-term potentiation is associated with increased [3H]AMPA binding in rat hippocampus.

The location and nature of the changes underlying long-term potentiation (LTP) remain controversial issues. In this study, we tested the possibility that changes in binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA/quisqualate and N-methyl-D-aspartate (NMDA) subtype of glutamate receptors are associated with LTP. LTP was elicited in vivo by stimulation of the perforant pathway in anesthetized rats. One hour following stimulation the animals were sacrificed. We performed quantitative ligand binding autoradiography on frozen brain sections using [3H]AMPA and [3H]N-(1-(2-thienyl)cyclohexyl)-3,4-piperidine ([3H]TCP) to label the AMPA/quisqualate and the NMDA receptors, respectively. No changes in [3H]TCP binding were detected in any of the treatment groups. However, increases in [3H]AMPA binding were observed only in animals that exhibited LTP. These increases were bilateral and present in several subfields of the hippocampus and cortical areas. Administration of the NMDA receptor antagonist, ketamine, prior to tetanic stimulation prevented both the increase in binding and the induction of LTP. These results suggest that changes in the characteristics of AMPA/quisqualate receptors are a biochemical correlate of LTP.


Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration.

Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration were investigated in the mouse sciatic nerves after nerve-transection at 3, 10 and 60 days of age, corresponding to the periods of early myelination, active myelination and post-myelination. As assessed by thymidine incorporation for the first 24 h in culture, Schwann cells from adult nerve proliferated rapidly within day 1 post-transection and reached a peak at day 3. In the nerves from neonatal or suckling mice, however, division rate of Schwann cells declined after transection, and was even less in the transected nerves than in the contralateral uninjured nerves. The reduction in thymidine uptake by Schwann cells was more pronounced in nerves sectioned at postnatal day 3 than those sectioned at day 10. By contrast, fibroblasts divided rapidly following transection regardless of age. These data suggest that mitogens from myelin components are important for proliferation of Schwann cells and that in the degenerating nerves of young mice, mitotic capacity of Schwann cells declined due to not only a loss of axonal mitogens but also the paucity of mitogens from myelin components. Proliferation of fibroblasts is likely to be stimulated by more general growth-promoting polypeptides common to any other tissues during wound repair.


Evidence for a substance P containing subpopulation in the primate suprachiasmatic nucleus.

Immunohistochemical detection of substance P (SP) in the suprachiasmatic nucleus (SCN) of the Old World monkey, Macaca fascicularis, was performed using two different rabbit polyclonal antisera. Immunostaining revealed a large population of neurons located in the dorsal subdivision of the nucleus identified by Nissl stain. This neuronal group represents the only cluster of SP-like immunoreactive (SP-IR) perikarya observed within the hypothalamus. In contrast with our present finding in the macaque, earlier studies only reported a few scattered SP-IR neurons in the SCN of other mammalian species. In agreement with previous descriptions of neuropeptides in the SCN, the topographical distribution of SP-IR neurons in the monkey confirms that cellular segregation is a significant feature of the mammalian SCN. This particular peptidergic subpopulation may represent a characteristic of the monkey circadian pacemaker. Together with other anatomical data previously reported in monkey and man, this finding also relates to the anatomical evolution of the circadian system from non-primates to humans. Although convincing data support the implication of SP in cyclic neuroendocrine regulations, the role of this tachykinin in circadian rhythmicity remains to be elucidated.


The segmental distribution of afferent fibers from the vaginal cervix and hypogastric nerve in rats.

Injections of horseradish peroxidase-wheat germ agglutinin (HRP-WGA) into the walls of the vagina and cervix (vaginocervical injections) of rats resulted in labeling of dorsal root ganglia (DRG) cells located at T11-L4 and L6-S2. In a second group of animals, exposure of the hypogastric nerve to HRP-WGA resulted in a similar bimodal distribution of labeled cells as compared to vaginocervical injections. In a third group, unilateral hypogastric nerve transection prior to injection of HRP-WGA into the vaginocervical walls resulted in a significant reduction in DRG cells labeled at T13, L1, L2, L6 and S1. Bilateral transection of the hypogastric nerves prior to vaginocervical injections eliminated labeled DRG cells at thoracolumbar levels but not at L6 and S1. Bilateral pelvic neurectomy reduced, but did not eliminate labeled DRG cells at L6 and S1 following vaginocervical injections. These results indicate that the hypogastric nerve constitutes a major sensory pathway from the vaginocervical walls to thoracic, lumbar and sacral levels of the spinal cord. The hypogastric nerve may subserve the transmission of noxious input from the vaginocervical walls as well as the activation of ascending spinal pathways involved in neuroendocrine reflexes during parturition.


N-methyl-D-aspartate (NMDA) and opioid receptors mediate dynorphin-induced spinal cord injury: behavioral and histological studies.

Both N-methyl-D-aspartate (NMDA) and opioid receptors have been implicated in the pathophysiology of traumatic spinal cord injury and dynorphin-induced paralysis. The present studies compared the effects of the non-competitive NMDA antagonist dextrorphan (Dex) and the kappa-selective opioid antagonist nor-binaltorphimine (nor-BNI) on the acute motor deficits and chronic neuropathological alterations caused by intrathecally administered dynorphin A-(1-17) (Dyn A). Infusion of Dyn A into the rat lower thoracic spinal subarachnoid space produced acute, reversible hindlimb paresis. Histological evaluations of spinal cord sections from these animals at 2 weeks post-infusion revealed ventral grey matter necrosis, neuronal loss and gliosis as well as axonal loss in adjacent white matter; however, there was minimal alteration in serotonin immunocytochemistry caudal to the injury zone. Dex or non-BNI pretreatment each significantly (P less than 0.05) reduced, and to a similar degree, the acute motor deficits and certain histological changes associated with Dyn A administration. These findings further support the hypothesis that dynorphin-induced spinal cord injury involves both NMDA receptors and opioid receptors.


Intraseptal connections redefined: lack of a lateral septum to medial septum path.

The integrity of the septohippocampal system is essential for memory formation and spatial behavior as well as for the electrical stability of the hippocampus. For many years it has been tacitly assumed or explicitly stated that the reciprocal septohippocampal loop is closed by a massive lateral septum-medial septum path. In the present study we reexamined the intraseptal connectivity with Phaseolus vulgaris leucoagglutinin tracing combined with choline acetyltransferase and parvalbumin immunohistochemistry at both the light and electron microscopic levels. We found that the previously hypothesized lateral septum to medial septum projection is extremely sparse and that the major medial septum to lateral septum path is parvalbumin-immunoreactive (likely GABAergic). The redefined circuitry has important implications for the understanding of the septal regulation of hippocampal electrical activity and the operations of the septo-hippocampal system.


Presence of GABA-immunoreactive neurons within intracortical patches in area 18 of the cat.

In cat visual cortex, horizontal, intracortical connections spread laterally to link together specific columnar sites. When visualized by retrograde tracers, these intracortical connections appear as periodic, columnar patches of dense cellular labeling interspersed with areas of much less dense labeling. We looked for anatomical evidence for direct inhibition among the patchy, horizontal connections in area 18, by combining retrograde labeling using wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP) with immunohistochemistry using an antiserum against the inhibitory neurotransmitter gamma-amino butyric acid (GABA). We found numerous double-labeled cells associated with some, but not all, of the local patches nearest to the injection site. In the superficial layers, the GABA-immunoreactive cells also labeled with WGA-HRP were confined to a zone approximately 1.0 mm from the center of the injection, while the double-labeled cells in the deeper layers spanned greater distances, up to 3.0 mm from the injection center. These more distant, double-labeled cells in the deeper layers were located on the edges or outside of the patches of dense labeling. Thus, all of the more distant intracortical patches, as well as some of the more proximal patches were devoid of double-labeled cells--a finding which suggests that direct inhibition may occur among only a selected group of the 'short range' intracortical patches and among none of the long-range patches.


Stimulation of inositol phosphate formation in cultured human retinal pigment epithelium.

Several hormones, neurotransmitters, and neuropeptides were screened for the ability to stimulate inositol phosphate formation in cultured human retinal epithelial (RPE) cells. Carbachol, vasopressin and thrombin were found to be effective. Treatment of RPE cells with all three agents produced increases in inositol monophosphate, inositol bisphosphate and inositol trisphosphate in the presence of 10 mM LiCl. Carbachol stimulated a 4-fold increase in the total of inositol phosphates at 1 mM. Studies with cholinergic antagonists showed a rank order of 4 DAMP greater than QNX greater than pirenzepine greater than methoctramine, suggesting the presence of M3 muscarinic receptors. Vasopressin gave a 2.5-fold stimulation at 10 microM. Agonists of vasopressin were also tested and gave differential responses. Studies using a V1 agonist (PIOVP) and a V2 agonist (DAVP) showed DAVP matching the level of stimulation elicited by vasopressin whereas treatment with PIOVP only reached 50% of the vasopressin response. These data suggested the presence of V2 receptors in the RPE cells. Several proteases were tested for their ability to stimulate RPE inositol phosphates. Thrombin caused a 7-fold increase in inositol phosphate formation at 1 U/ml, whereas trypsin and plasmin elicited smaller responses (approximately 2-fold). The thrombin effect was blocked by the thrombin-specific inhibitor, hirudin, but not by other protease inhibitors. Several mediators of inflammation such as bradykinin, histamine and serotonin were also tested, and they were ineffective in stimulating inositol phosphate turnover in the RPE cells.


Agents that affect calcium influx can change cyclic nucleotide levels in cultured chick pineal cells.

Previous experiments examined interactions among the effects of cyclic AMP and calcium-related agents on melatonin output by cultured chick pineal cells, and suggested that changes in calcium influx might act through cyclic AMP. Here, effects of calcium-related agents and manipulations on cyclic AMP (and cyclic GMP) levels are demonstrated directly. These effects support a role for cyclic AMP (but not cyclic GMP) in the effects of changes in calcium influx on melatonin production by these cells.


Immunohistochemical characterization of NPY and substance P containing nerve terminals in aged and diabetic human sympathetic ganglia.

To compare the neuropeptide specificity of dystrophic axon formation in aging versus diabetic human sympathetic ganglia we have immunohistochemically characterized neuropeptide Y (NPY) and substance P containing intraganglionic nerve terminals. Prevertebral superior mesenteric but not paravertebral superior cervical ganglia developed markedly swollen NPY containing axonal termini with both aging and diabetes. Substance P containing nerve terminals failed to develop dystrophic changes. Selective loss of classes of nerve terminals may result in discrete functional sequellae.


gamma-Aminobutyric acid, through GABAA receptors, inhibits the potassium-stimulated release of calcitonin gene-related peptide- but not that of substance P-like material from rat spinal cord slices.

Superfusion of slices of the dorsal zone of the lumbar enlargement with an artificial cerebrospinal fluid was used to investigate the possible modulation by GABA receptor ligands of the in vitro release of calcitonin gene-related peptide- and substance P-like materials (CGRPLM and SPLM) from the rat spinal cord. Whereas the spontaneous outflow of both peptides remained unaffected, the K+ (30 mM)-evoked overflow of CGRPLM could be partially inhibited (approx. -30%) by GABA (1 microM-0.1 mM) and muscimol (10 microM-0.1 mM) but not by baclofen (1-10 microM). Bicuculline methiodide (1 microM) completely prevented the inhibition by GABA (1 microM) and muscimol (10 microM) as expected from an action through GABAA receptors. By contrast, the K(+)-evoked SPLM overflow was altered neither by GABA nor muscimol and baclofen. These data further support that GABA exerts a presynaptic inhibitory control of (CGRP-containing) primary afferent fibres within the rat dorsal horn.


Distribution of bulbospinal neurons supplying bilateral innervation to the phrenic nucleus in the rat.

The location of bulbospinal neurons with axon collaterals in both phrenic nuclei were determined by injecting two different fluorescent tracers into the right and left C4 cervical spinal cord. In contrast to single-labeled neurons that were found throughout the rostrocaudal extent of the medulla, the majority of double-labeled neurons were located in the rostral ventral respiratory group. Only a few double-labeled neurons were found in the ventrolateral nucleus of the solitary tract. The role of this bilateral pathway in synchronizing the activity of the phrenic nucleus is discussed.


Susceptibility of brain to AMPA induced excitotoxicity transiently peaks during early postnatal development.

The excitatory and excitotoxic actions of the endogenous excitatory amino acid (EAA) neurotransmitter, glutamate, are mediated by activation of three common subtypes of EAA receptors: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/quisqualate and kainate receptors. EAA neurotransmitter systems play a number of physiological roles in the regulation and organization of neural systems during development. However, excessive activation of this neurotransmitter system is also implicated in the pathophysiology of several forms of acute and chronic brain injury. In this study, the susceptibility of the developing rat brain to AMPA/quisqualate receptor mediated injury was examined at eight postnatal ages (1-90 days). The receptor agonists, AMPA (25 nmol) or quisqualate (100 nmol), were stereotaxically microinjected unilaterally into the anterior striatum. The severity of resulting brain injury was assessed 5 days later by comparison of reductions in regional cortical and striatal cross-sectional areas. Microinjection of AMPA (25 nmol) produced widespread unilateral forebrain injury in the intermediate postnatal period (days 5-28). The severity of injury resulting from microinjection of a fixed dose of AMPA (25 nmol) transiently exceeded the severity of injury in adults between PND 5-28 with peak sensitivity occurring near PND 10. At PND 1, microinjection of AMPA produced a 24.5 +/- 1.7% reduction in striatal cross-sectional area, which is similar to the response observed in adult animals, and the lesion was confined to the injection site. Susceptibility to AMPA toxicity increased 2-fold from PND 1 to PND 5. At PND 10, the age of maximal sensitivity, the excitotoxic reaction to AMPA extended throughout the entire cerebral hemisphere and the mean striatal cross-sectional area was reduced by 81.7 +/- 3.9%. With advancing postnatal age, the severity of injury progressively diminished and the lesion became confined to the injection site. The developmental pattern of sensitivity to AMPA toxicity in other brain regions differed although peak sensitivity consistently occurred near PND 10. Microinjection of quisqualate produced a developmental pattern of striatal susceptibility similar to AMPA although quisqualate was a considerable less potent neurotoxin. In additional experiments, the in vivo pharmacology of AMPA and quisqualate mediated brain injury was evaluated in a PND 7 rat model in order to determine the neurotoxic characteristics and specificity of these agonists in vivo. The severity of brain injury was assessed 5 days after intrastriatal excitotoxin injection by comparison of cerebral hemisphere weights.(ABSTRACT TRUNCATED AT 400 WORDS)


Dopaminergic neuron development in rats: biochemical study from prenatal life to adulthood.

Dopaminergic cell development has been studied mainly using morphological techniques and especially histofluorescence. However, the biochemical characteristics of dopamine (DA) neuron development and its physiological role during ontogeny are much less known. In the present article, the biochemical development of DA neurons, from day 13 of prenatal life to adulthood, is evaluated in Sprague-Dawley rats. DA was first detected on day 14 of gestation. The brain increase in this neurotransmitter begins on day 17 in the proencephalon and on day 18 in the mesencephalon, reaching on day 20 a level similar to that found during adulthood in the latter but not in the former. DA levels in the proencephalon rise slowly to adulthood level when compared to DA in the mesencephalon. The modifications observed in tyrosine levels are also largely similar to those reported for DA. Finally, the study of the first 48 h of life shows an increase in tyrosine levels and a decrease in dihydroxyphenylacetic acid levels (with a reduction of DA turnover) during the first 4-5 h of postnatal life. Since the serotonergic modification was completely different from DA modification, we conclude that the biochemical alteration of DA neurons during early postnatal development is specific. The present data suggest that DA neurons play different roles before and after reaching adult development.


The germ cell-less gene product: a posteriorly localized component necessary for germ cell development in Drosophila.

The first cell fate specification process in the Drosophila embryo, formation of the germline precursors, requires posteriorly localized germ plasm. We have cloned a gene, germ cell-less (gcl), required for germline formation. Posterior localization of the gcl messenger RNA (mRNA) requires the function of those genes essential for the localization of both nanos RNA, which specifies the abdomen, and the germ cell determinants. Mothers with reduced gcl function give rise to sterile adult progeny that lack germ cells. In embryos with reduced maternal gcl product, the germ cell precursors fail to form properly. Consistent with this phenotype, gcl protein specifically associates with those nuclei that later become the nuclei of the germ cell precursors. These observations suggest that gcl functions in the germ cell specification pathway.


Biochemical modulation of 5-fluorouracil with or without leucovorin by a low dose of brequinar in MGH-U1 cells.

Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical anti-tumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU +/- leucovorin (LV) +/- brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC50) for brequinar, FU, and FU+LV (100 microM) were 0.4, 20, and 10 microM, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC10 (0.1 microM) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 microM brequinar and FU +/- LV did not increase the cytotoxicity of FU +/- LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 microM brequinar for 24 h alone or followed by a 2-h exposure to FU (25 microM) +/- LV (100 microM). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU+brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 microM hypoxanthine and 25 microM FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).


In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli.

Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.


In vivo effects of doxorubicin on kinase C in cultured cells.

The ability of doxorubicin to inhibit kinase C in vivo in cultured BALB/c 3T3 mouse fibroblasts was determined by the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and the induction of c-fos transcription following treatment with 200 nM phorbol 12-myristate 13-acetate. A concentration of 60 microM doxorubicin did not inhibit MARCKS phosphorylation but completely inhibited c-fos expression. The inhibition of c-fos expression was not specifically mediated via kinase C, since both the total RNA synthesis as measured by [3H]-uridine uptake and the fraction of serum-induced c-fos expression that was not attributable to kinase C were inhibited by doxorubicin. The present data do not support the hypothesis that the cytotoxic effect of doxorubicin is attributable to the inhibition of kinase C in vivo.


Tc-99m HMPAO WBC imaging to detect carditis and to evaluate the results of high-dose gamma globulin treatment in Kawasaki disease.

Eighteen patients with Kawasaki disease and suspected carditis (11 boys, 7 girls, mean age 18 months) in acute stages underwent Tc-99m HMPAO WBC imaging of the heart. Signs and symptoms subsided after conventional aspirin therapy and the intravenous injection of high-dose (400 mg/kg per day for 5 days) gamma-globulin treatments. Tc-99m HMPAO WBC imaging of the heart was arranged a second time to evaluate the effects of the treatments for carditis. The results showed that a significant difference existed in the severity of carditis before and after the treatments (P less than 0.001, by signed rank test), 39% (7/18) with significant improvement in severity of carditis, 50% (9/18) without definite change. However, 11% (2/18) became more severely ill after the treatments. Tc-99m HMPAO WBC imaging may be useful in detecting carditis in Kawasaki disease and in evaluating the effects of aspirin plus the newly recommended gamma-globulin for the treatment of carditis in Kawasaki disease.


Ca2+/calmodulin-regulated nitric oxide synthases.

NO synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and nitric oxide (NO) or a NO-releasing compound. At least three isoforms of NOS exist (types I-III). The activities of the type I isoform purified from brain and the type III isoform purified from endothelial cells are regulated by the intracellular free calcium concentration ([Ca2+]i) and the Ca(2+)-binding protein calmodulin. At resting [Ca2+]i, both isozymes are inactive; they become fully active at [Ca2+]i greater than or equal to 500 nM Ca2+. Longer lasting increases in [Ca2+]i may downregulate NO formation, for in vitro phosphorylation by Ca2+/calmodulin protein kinase II decreases the Vmax of NOS. Besides the conversion of L-arginine, type I NOS, Ca2+/calmodulin dependently, generates H2O2 and reduces cytochrome c/P450. Other redox activities, i.e. the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by NOS appear to be Ca2+/calmodulin-independent.


Epoetin enhances erythropoiesis in normal men undergoing repeated phlebotomies.

Epoetin may enhance autologous blood donation, but efficacy and dose response have not been established. This multicenter, double-blind trial compared intravenous placebo (n = 23) with epoetin beta, 250 U/kg (n = 23), 500 U/kg (n = 19), and 1000 U/kg (n = 22), administered three times weekly for 26 days. Normal men (age, 28 +/- 7 years; mean +/- SD) received phlebotomies up to three times weekly as long as the hemoglobin remained greater than or equal to 12 gm/dl. Subjects treated with epoetin donated 32% more units of blood (p less than 0.05) compared with placebo. A dose response was not observed. Platelet counts increased with epoetin compared with placebo, but platelet function and bleeding time did not change. Prothrombin times increased and partial thromboplastin times decreased with both epoetin and placebo. The supernatant of packed red blood cells collected after multiple phlebotomies and stored 42 days had slightly lower glucose concentrations and pH after therapy with epoetin. Blood pressure did not change with epoetin or placebo. These findings support the efficacy and safety of epoetin for enhancing the erythropoietic response of normal subjects during intensive phlebotomy.


A member of the selectin family (GMP-140/PADGEM) is expressed on thrombin-stimulated rat platelets in vitro.

1. Granule membrane protein (GMP-140) is an integral alpha-granule membrane glycoprotein, expressed on the surface of human platelets following degranulation, and is part of a new family of adhesion molecules (selectins) related to the endothelial leukocyte adhesion molecule (ELAM-1) and to the lymphocyte homing receptors in man (Leu-8/TQ1) and in mouse (gp90MEL-14). 2. The cross-reactivity with rat platelets of the monoclonal antibodies (MAb), LYP20 and S12, directed against human GMP-140 was examined, with the purpose of assessing the homology of GMP-140 between human and rat platelets and of using positive MAbs to detect platelet activation in vivo in response to vascular disease in rats. 3. By ELISA technique, LYP20 gave a greater OD reading with thrombin-stimulated rat platelets than with resting platelets. 4. 125I-LYP20 bound significantly more to thrombin-stimulated rat platelets (3875 +/- 750 molecules/platelet) than to resting platelets (645 +/- 240 molecules/platelet, P less than 0.01) with 50% maximum binding at 0.13 +/- 0.02 microgram/ml; 125I-S12 did not bind to rat platelets. 5. By fluorescence-activated flow cytometry there were significantly more fluorescent thrombin-stimulated platelets (56 +/- 7% of total), compared with resting platelets (8 +/- 1% of total, P less than 0.001). 6. Western blots of rat platelet lysates showed that LYP20 bound to a single band identified, under non-reducing conditions, as having the same apparent M(r) as GMP-140. 7. LYP20 immunoprecipitated a protein which became radiolabelled on the surface of thrombin-activated rat platelets; S12 did not recognize any protein.(ABSTRACT TRUNCATED AT 250 WORDS)


Vasoactive intestinal polypeptide-containing nerve fibers are increased in abundance in the choroid of dystrophic RCS rats.

As photoreceptor degeneration progresses in Royal College of Surgeons (RCS) rats, a variety of morphological and physiological alterations occur in the outer retina. Since the choriocapillaris responds to changes in the outer retina in other retinopathies, we examined the possibility that changes in the choroidal vasculature also occur in RCS rats. The choroidal and choriocapillary vessels in RCS and control (RCS-rdy+) rats were examined during the period after which photoreceptor loss and retinal vascular changes had occurred (7-mos to 28-mos). Light microscopic (LM) morphometry and electron microscopic (EM) examination showed no significant differences between these groups in the number, size or morphology of these vessels. However, EM image analysis revealed that nerve fibers and bundles were twice as abundant in the RCS choroid than in the control. Using immunohistochemical techniques at the LM level combined with image analysis we found that vasoactive intestinal polypeptide positive (VIP+) fibers were significantly increased in the RCS choroid compared with control choroid. In contrast, the abundance of immunoreactive fibers labelled for substance P and dopamine beta hydroxylase appeared similar in both the control and RCS choroid. Since VIP is a potent vasodilator, the increased abundance of nerve fibers in the RCS choroid in conjunction with the unaltered number and size of these vessels suggests that choroidal blood flow may be increased. It is uncertain whether this increase is a response to the outer retinal pathology or contributes to it.


Localization of the lysosomal protease dipeptidyl peptidase II in the young normal rat lens: a correlative light and electron microscopic analysis.

This investigation was follow-up to an earlier biochemical and light microscopic histochemical study, in which the lysosomal protease dipeptidyl peptidase II (DPP II) was demonstrated in rodent lenses. In the present study, a method was employed that allowed a more precise histochemical localization of the enzyme, one that was suitable for ultrastructural as well as light microscopic analysis. Successful demonstration of the enzyme using either of two synthetic substrates, and the significant reduction of the enzyme reaction by phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor, pointed to the sensitivity of the method. A flat-embedding technique allowed the correlative light and electron microscopic analysis of specific areas of the specimen. Examination of the epithelium and outer cortical regions of the lens revealed the compartmentalization of DPP II activity within lysosomal dense bodies that were concentrated primarily in the equatorial and sutural regions, and also an association of the reaction product with larger bodies that were confined to the sutural regions. The latter structures appeared to represent fiber cell fragments that were enwrapped with narrow extensions of the surrounding fiber cells. The location of enzyme activity within the sutural bodies and also within the intercellular spaces of the modified fiber cell extensions surrounding these bodies suggested that lysosomal proteases may play a role in the segregation and degradation of specific regions of normal lens fiber cells.


Cytogenetics of bisexual/unisexual species of Poecilia. II. Analysis of heterochromatin and nucleolar organizer regions in Poecilia mexicana mexicana by C-banding and DAPI, quinacrine, chromomycin A3, and silver staining.

Chromosomes of Poecilia mexicana mexicana, one of the bisexual species involved in the hybrid origin of the unisexual teleost fish species P. formosa, were analyzed by several staining techniques. Sex-specific, differential heterochromatin, found in other congeneric species, was not observed in P. m. mexicana. Nucleolar organizer regions were polymorphic among individual specimens within a given population sample. A single specimen exhibiting intraindividual variability of chromosome pair 1 and a specimen with a triploid karyotype are also described.


Localization by in situ hybridization of a type I keratin intermediate filament gene (Krt-1.14) to band D of mouse chromosome 11.

A probe from the 3' noncoding region of a murine type I keratin intermediate filament (IF) gene (Krt-1.14) localizes to band D of murine Chromosome 11 using in situ hybridization. This localization provides a physical confirmation of the assignment of the type I keratin genes by linkage analysis in the mouse. It also demonstrates that the Krt-1.14 genes are at a single locality in the mouse in contrast to the two locations on the short and long arms of chromosome 17 in humans.


Localization of four human chromosome 21 genes--SOD1, ETS2, IFNAR, and CBR--to two different chromosomes in the marsupial species Macropus eugenii.

We have mapped the chromosomal location of four genes previously assigned to human chromosome 21--Cu/Zn superoxide dismutase (SOD1), the protooncogene ETS2, the interferon alpha/beta receptor gene (IFNAR), and the carbonyl reductase gene (CBR)--in the tammar, Macropus eugenii. The genes are localized on two separate autosomes: SOD1 and CBR map to chromosome 7 and ETS2 and IFNAR map to chromosome 3 or 4. These results provide the first example of asynteny between SOD1/CBR and ETS2/IFNAR in a mammalian species. The results suggest that either this synteny group has been disrupted in the marsupial lineage, or, alternatively, the genes located on human chromosome 21 may have been joined after the marsupials diverged from the eutherian mammals some 130-150 million years ago.


[Free intraperitoneal tumors cells in pancreatic cancer--significance for clinical course and therapy].

Intraoperative peritoneal cytology was performed in 36 patients with pancreatic ductal adenocarcinoma. 12 patients (33%) showed malignant cells in the peritoneal cavity. In the further course these patients developed more non-local metastases and had a significantly shorter survival rate. Peritoneal carcinomatosis became evident in 75% of the patients with free cancer cells in contrast to only 14% of the patients without. Detection of free cancer cells was directly related to the histological tumor stage (TNM-system). Iatrogenic shedding of malignant cells by surgical tumor manipulation or needle biopsy was not observed. The technique of intraoperative peritoneal lavage with consecutive cytology and its possible effects on further treatment is discussed.


[Hemodynamic surveillance in pregnancy induced hypertension during volume expansion therapy].

Hemodynamic parameters were studied in 80 cases of pregnancy induced hypertension (PIH) using noninvasive cardiovascular detector (TP-CBS). Women were categorized into 3 kinds of hemodynamic patterns: (1) normal cardiac output, 45 cases, cardiac index (CI) = 2.5-4 L.min-1/m2; (2) high cardiac output, 10 cases, CI greater than 4 L.min-1/m2; (3) low cardiac output, CI less than 4 L.min-1/m2, 25 cases (31.5%). Ten cases in each category were selected for test after volume expansion therapy. MAP decreased in varying degrees. CI showed marked increase in the low cardiac output group, but decreased to normal in the high output group. The monitoring criteria for protecting against pulmonary edema during volume expansion therapy were discussed.


CD5+ B cells are decreased in peripheral blood of patients with Crohn's disease.

B cells bearing the CD5 surface marker comprise a substantial minority of the circulating lymphocyte population in healthy individuals. These recently described cells have been implicated in T-independent humoral responses, immunoregulation, and autoimmunity. We undertook to enumerate circulating CD5+ B cells by three-color fluorescence activated flow cytometry in 28 patients with Crohn's disease (CD). None of the CD patients were using immunosuppressive medication. The CD patients were subdivided into "inactive" and "active" groups based upon their Crohn's disease activity index (CDAI). Thirty-two normal subjects served as a control population. The percentage of CD19+ B cells was significantly reduced in both active and inactive CD patients as compared with normal controls (P less than or equal to 0.01). CD5+ B cells were likewise found to be significantly decreased in both inactive and active CD patients (P less than or equal to 0.01) as compared with normal controls. The proportion of CD5+ B cells was significantly lower in the peripheral blood of active as compared with inactive CD patients (P less than or equal to 0.05). The finding that CD5+ B cells are reduced in CD may provide an important clue to immunological dysfunction in inflammatory bowel disease and merits further study.


Role of ischemia in acute pancreatitis. Hemorrhagic shock converts edematous pancreatitis to hemorrhagic pancreatitis in rats.

Ischemia has been considered to play a role in the development of acute pancreatitis. The aim of this study was to investigate the effect of ischemia, caused by hemorrhagic shock, on cerulein-induced acute pancreatitis in rats. Acute pancreatitis was induced by the intravenous infusion of a supramaximally stimulating dose of cerulein (10 micrograms/kg/hr) for 6 hr. Hemorrhagic shock was induced by the removal of blood until the mean arterial blood pressure reached 35 mm Hg. This level was maintained for 30 min, after which time all the blood was reinfused. Hemorrhagic shock alone induced no morphological change in the pancreas. However, after the induction of hemorrhagic shock in animals treated with cerulein, hemorrhage and parenchymal necrosis were frequently observed in the pancreas. Seven of 20 rats (35%) receiving cerulein plus hemorrhagic shock had died by 48 hr after the start of cerulein infusion, whereas none of the rats in the cerulein or shock group died during this experiment. Cathepsin B activity in the pancreas of the cerulein plus shock group was significantly higher than in the other groups at 48 hr. These results suggest that ischemia may be a contributing factor in the pathogenesis of acute pancreatitis.


Histopathologic correlates of serum amylase activity in acute experimental pancreatitis.

The association of serum amylase activity with the extent of pancreatic injury in acute pancreatitis is unclear. To clarify this relationship, we induced acute pancreatitis ranging from mild to lethal in 118 Sprague-Dawley rats (350-450 g). This was achieved by controlled intraductal infusion of low- or high-dose bile salt, with or without enterokinase, followed by intravenous cerulein or saline for 6 hr. Serum amylase was measured at baseline and 6 hr. Pancreatic histopathology was evaluated by two blinded pathologists employing total surface scoring (N = 118) and morphometric 20-field documentation (N = 22). Serum amylase correlated best with edema (r = 0.61) and fat necrosis (r = 0.58), less well with acinar necrosis (r = 0.53) and inflammation (r = 0.50), and poorly with hemorrhage (r = 0.33) and perivascular infiltrate (r = 0.31). Inasmuch as edema and fat necrosis are not important determinants of severity, these observations could explain the poor prognostic value of serum amylase activity in patients with acute pancreatitis.


Effects of long-acting somatostatin analog (SMS 201-995) on eicosanoid synthesis and survival in rats with acute necrotizing pancreatitis.

The effects of a long-acting somatostatin analog (SMS 201-995) were studied in an established model of acute necrotizing pancreatitis in rats. SMS 201-995, when given prior to induction of pancreatitis, decreased the mortality rate from 100% to 40% (P = 0.0001). When treatment was given after induction of pancreatitis, the mortality rate was 75% (P = 0.2). Administration of SMS 201-995 did not influence the serum concentrations of amylase markedly, but the lipase levels were significantly lowered (P less than 0.05). The low levels of serum insulin and the glucose level in whole blood were not influenced. The volume of ascitic fluid was reduced (P less than 0.01). Moreover, less peritoneal fat necrosis was seen, suggesting a reduction in toxic factors in the ascitic fluid. Treatment with SMS 201-995 prior to induction of pancreatitis caused a significant increase in the levels of circulating 6-keto-PGF1 alpha, the stable metabolite of prostaglandin I2 (P less than 0.01). The levels of thromboxane B2 and prostaglandin E2 did not change significantly. The present data support the hypothesis that SMS 201-995 is an activator of prostaglandin I2, thereby modifying the course of the disease.


Methodology of antiemetic trials.

Progress in antiemetic research dictates that clinical trials of antiemetic agents be conducted according to guidelines for Good Clinical Practice, as follows. Studies must be of a prospective, parallel-group design in which the new treatment is compared with the existing best available treatment, after optimal dosage schedules for each have been established. Ethically, placebo-controlled trials can only be justified when chemotherapy with a low emetogenic potential is used. All end-points (nausea, vomiting, adverse events and quality of life parameters) must be specified in detail before the trial is begun. Patient populations must be homogenous with respect to prior chemotherapy and other confounding variables. Finally, patients must actively participate in the evaluation of antiemetic therapy, since only they can provide reliable information regarding the impact of nausea and vomiting on their quality of life.


Tropisetron. A review of the clinical experience.

This review describes clinical experience with tropisetron, a new 5-hydroxytryptamine type 3 (serotonin 3)-receptor antagonist, which was found to possess antiemetic properties in animal studies and pilot studies in chemotherapy-treated patients. Tropisetron 5mg once daily is an effective and well tolerated antiemetic treatment for chemotherapy-induced emesis. Tropisetron can be administered without special precautions to all patients who undergo aggressive chemotherapy, and remains effective during multiple chemotherapy courses. The efficacy of tropisetron compares well with that of the best available complicated antiemetic cocktails, but tropisetron is better tolerated. The simple dosage schedule of either 1 injection or 1 capsule per day makes tropisetron ideal for both inpatient and outpatient treatment.


Prevention of chemotherapy-induced nausea and emesis in patients responding poorly to previous antiemetic therapy. Comparing tropisetron with optimised standard antiemetic therapy.

In a multicentre trial, 78 patients with a variety of malignancies, who had experienced insufficient control of emesis (greater than or equal to 3 episodes within 24 hours) while receiving standard antiemetics during previous chemotherapy, were randomly assigned to receive tropisetron 5mg once daily for 5 days or conventional antiemetic drugs. No attempt was made to standardise the conventional antiemetic treatment, which was given according to the usual practice of the participating institutions. Emesis was evaluated by counting emetic episodes and nausea by asking the patients to record on a diary chart the duration and severity of the nausea. Emesis was much better controlled with tropisetron than with standard drugs, complete control during the first 24 hours being achieved in 42% and 8% of patients, respectively, (p less than 0.001). Nausea was of significantly shorter duration (6.9 vs 10.3 hours; p less than 0.01) and was less severe (p less than 0.005) in the tropisetron group. The patients' overall assessment of treatment outcome was markedly better for tropisetron than for the standard antiemetic therapy. The superior efficacy of tropisetron was especially marked during the first 24 hours. For delayed nausea, no significant difference between treatments was seen. No serious adverse effects were observed.


Compassionate use of a 5-HT3-receptor antagonist, tropisetron, in patients refractory to standard antiemetic treatment.

The efficacy of tropisetron in the prevention of nausea and vomiting induced by chemotherapy of varying emetogenic potential was evaluated in 545 patients with a variety of malignancies who had either proved refractory to antiemetic treatment during previous chemotherapy courses or who were considered to be at high risk of nausea and vomiting. Tropisetron 5 or 10mg was administered intravenously just before chemotherapy, with the possibility of additional oral or intravenous doses on the day before chemotherapy and on 1 or more subsequent days. On day 1 of the first course of chemotherapy, a complete response (no nausea and no vomiting) was achieved in 62% of patients and a partial response (1 to 4 vomits and/or episodes of nausea) in 29%. Among the 325 patients who received a second course of chemotherapy, more than 80% of those with a complete response on day 1 of course 1 also had a complete response on day 1 of course 2; 37% and 26%, respectively, of patients with a partial response or failure (1 or more vomits and/or episodes of nausea) on day 1 of course 1 then had a complete response on day 1 of course 2.


Tropisetron, a new 5-HT3-receptor antagonist, in the prevention of radiation-induced nausea, vomiting and diarrhoea.

Oral tropisetron, a 5-hydroxytryptamine type 3 (serotonin3) [5-HT3]-receptor antagonist, at a dose of 5mg daily was evaluated as antiemetic prophylaxis during postoperative abdominal irradiation. 20 women with International Federation of Gynecology and Obstetrics (FIGO) stage I to III ovarian carcinoma were included. 12 women received irradiation of whole abdominal fields and 8 of lower abdominal/pelvic fields. Efficacy and adverse events were recorded by the patients in diary-form booklets. The cumulative weekly incidence of patients with nausea, which was generally mild and of short duration, increased from 30% at the start of radiotherapy to 54% at the end of treatment. Episodes of vomiting occurred in less than 10% of the patients. Diarrhoea was common towards the end of the radiotherapy courses, and the proportion of patients needing extra antidiarrhoeal medication (loperamide) increased from 38% during the first week to 100% at the end of the radiotherapy course. Mean weight loss was 1.2kg during the 5- to 6-week course. Overall ratings for quality of life were excellent or good in 75 to 85% of patients. Tropisetron seems to be a promising and well tolerated drug in conjunction with extended radiotherapy of abdominal fields. This was an open study, establishing the methodology for long term follow-up of patients during fractionated radiotherapy.


Three years' experience with tropisetron in the control of nausea and vomiting in cisplatin-treated patients.

The efficacy and tolerability of tropisetron in preventing cisplatin-induced nausea and vomiting was studied in 2 open trials and compared with the efficacy and tolerability of metoclopramide plus lorazepam in a randomised crossover trial. In the first study, tropisetron 10mg was administered intravenously over 15 minutes before the cisplatin infusion and a second 10mg dose was given after the 60-minute infusion of cisplatin (greater than 50 mg/m2) in 54 patients with advanced cancers, for a total of 165 courses. Good responses for nausea and vomiting were recorded in 83.0% and 87.9% of courses, respectively, with complete protection from nausea and vomiting in 44.8% and 66.1% of courses, respectively. In the second study in 25 patients whose characteristics and cisplatin schedule were comparable with those of the first study, very similar results were achieved in 104 courses of chemotherapy, despite a reduction in tropisetron dose to a single 5mg intravenous infusion 15 minutes before cisplatin. The efficacies of intravenous tropisetron 5mg and metoclopramide 2 mg/kg plus lorazepam administered 15 minutes before cisplatin in preventing acute and delayed nausea and vomiting were compared in a randomised crossover study involving 20 patients. Tropisetron was significantly superior (p less than 0.001) in controlling both acute and delayed (day 1) symptoms. In all studies, the tolerability of tropisetron was excellent. The most frequent side effect was mild to moderate headache, occurring in 5 to 7% of patients. In conclusion, our experience suggests that tropisetron is an effective and well tolerated antiemetic drug that improves the quality of life of cancer patients administered highly emetogenic chemotherapy regimens.


Biochemical analyses of proteolytic nicking of the human glycoprotein hormone alpha-subunit and its effect on conformational epitopes.

Conformational features of two epitopes on the glycoprotein hormone alpha-subunit were investigated using two antihuman FSH (anti-hFSH) monoclonal antibodies (mAbs) 3A and 5F that recognize different epitopes and are specific for alpha-subunit. These mAbs were used to investigate whether the conformation of these epitopes was different in heterodimeric hFSH, hTSH, hLH, or hCG. Any differences in the mass of hormone in each preparation were accounted for by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blot analysis of all hormone preparations used in this study. Rabbit anti-hFSH alpha-(11-27) antipeptide antisera and [125I]protein-G were used in the Western blot analysis. Radioactivity associated with each band was determined and used to normalize the mass of alpha-subunit in each reference preparation used in the displacement assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also performed in order to examine the integrity of each of the hormone reference preparations. hTSH alpha and, to a lesser extent, hLH alpha preparations contained an internal nick in the polypeptide chain. RIA analysis performed using heterodimeric glycoprotein hormones as competitors revealed that an average 100-fold difference in the ED50 values for hFSH compared to the other glycoprotein hormones was seen with mAb 3A. Therefore, the conformation of 3A epitope appeared to be different in hFSH than in hTSH, hLH, or hCG. In comparison, the epitope recognized by mAb 5F only had an average 7-fold difference in reactivity (ED50 values) for hFSH compared to hTSH, hLH, and hCG. Likewise, competition assays using the respective alpha-subunits and mAb 5F revealed a pattern of competition similar to that observed with heterodimers, with an average 4-fold difference in the ED50 values for hFSH alpha compared to those for hTSH alpha, hLH alpha, and hCG alpha. Therefore, the conformation of the 5F epitope appears unaffected by association of alpha-subunit with beta-subunit. Accordingly, any differences in the conformation of the four alpha-subunits, as demonstrated by these small differences in ED50 values, appear to be inherent to each alpha-subunit. In fact, the 5F epitope appears to be quite rigid, since nicked alpha-subunit preparations could compete with [125I]hFSH for binding to 5F with comparable potency to non-nicked alpha-subunits. These findings support the concept that epitopes on heterodimeric hFSH alpha may have different conformational features. Some are specific for heterodimeric hFSH alpha, and we refer to these as conformationally active (flexible). Others are common to the four human glycoprotein hormone alpha-subunits, suggesting that they are conformationally constrained (rigid).


Mitogenic activity of epidermal growth factor on newborn rat astroglia: interaction with insulin-like growth factors.

Newborn rat astroglia cells possess epidermal growth factor (EGF) and insulin-like growth factor (IGF) receptors, which suggests that these growth factors regulate their growth and development. To determine the relative roles and interactions between the two growth factors on astroglial growth, primary cultures of astroglial cells from newborn rats (1 day postnatal) were treated with pure peptides, singly or in combination in various concentrations, and the growth response was determined by DNA synthesis ([3H]thymidine incorporation). EGF, IGF-I, and IGF-II, as single peptides, stimulated DNA synthesis, with half-maximal stimulatory concentrations of 0.25 ng/ml for EGF, 2.0 ng/ml for IGF-I, and 25 ng/ml for IGF-II, respectively. These findings indicate that astroglial cells are responsive to these growth factors in physiological concentrations, with the relative sensitivity of EGF greater than IGF greater than IGF-II. When EGF and IGF-I were added in combination, the growth stimulatory effect was greater than the additive effects of each growth factor added alone, indicating that the two growth factors act in synergism with each other. In particular, addition of increasing concentrations of EGF from 0.25-10 ng/ml to a constant concentration of 50 ng/ml IGF-I resulted in significant potentiation of [3H]thymidine incorporation of astroglial cells. To determine if the synergistic effect was due to a local synthesis of IGF-I by astroglia, a specific monoclonal antibody against IGF-I (Sm 1.2) was added to the peptides. Sm 1.2 decreased not only IGF-I-stimulated DNA synthesis, but also EGF-stimulated DNA synthesis, suggesting that the effects of EGF were contributed to in part by the local synthesis of IGF-I by astroglial cells. Analysis of conditioned medium from cells treated with EGF revealed a significant increase (approximately 2-fold) in IGF-I (from 4.5 to 8.8 ng/ml), but not IGF-II. To determine if the EGF effect on IGF synthesis was at the level of IGF-I mRNA transcription, stable IGF-I mRNA levels were determined in the astroglial cells before and after stimulation with EGF, using Northern analysis and quantification by densitometry. Astroglia expressed four IGF-I mRNA transcripts as in the adult and fetal liver, but only one (3.6 kilobases) IGF-II mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)


Diverse effects of tachykinins on luteinizing hormone release in male rats: mechanism of action.

The tachykinins are a group of structurally related peptides found in the rat hypothalamus and anterior pituitary. We have evaluated the effects of four tachykinins on LH release in male rats. In intact male rats, intracerebroventricular (icv) injection of neurokinin A (NKA), neuropeptide K (NPK), and neuropeptide-gamma (NP gamma) elicited dose-related, transient increases in plasma LH. Substance P (SP) was ineffective under these conditions. A further examination showed that in vitro incubation with either NPK or NP gamma of hemipituitaries from intact but not castrated male rats promoted release of LH into the medium, thereby revealing that the excitatory effects of tachykinins in intact male rats may, in part, be a result of stimulation of LH release directly from the anterior pituitary. On the other hand, the effects of these four tachykinins on LH release were different in castrated rats. Intracerebroventricular injection of NPK, NKA, and NP gamma as well as SP, which was ineffective in intact male rats, evoked a long-lasting suppression of LH release. Comparatively, NPK was the most effective tachykinin in eliciting LH responses in both of these tests involving different endocrine environments. We next evaluated the possibility that the inhibitory effects of tachykinins (NPK) may be mediated by activation of inhibitory endogenous opioid peptides. The results showed that iv infusion of the opiate receptor antagonist naloxone, to block the possible inhibitory effects of endogenous opioid peptides, only partially counteracted the suppressive effects of icv NPK on plasma LH levels. Thus, in addition to revealing the diverse effects of structurally related tachykinins on LH release, the results of these investigations showed specifically that the NK-2 receptor agonists NPK, NP gamma, and NKA stimulated LH release in intact rats, in part, by a direct action at the level of the pituitary, whereas the NK-1 receptor agonist SP was inactive under these conditions. These findings imply a paracrine/autocrine mode of excitatory action on LH release involving pituitary NK-2 receptor subtypes. On the other hand, in castrated rats, all four tachykinins readily suppressed LH release by a central action involving, in part, an activation of hypothalamic opioid systems.


Effect of follicle-stimulating hormone on insulin-like growth factor-I-stimulated rat granulosa cell deoxyribonucleic acid synthesis.

Granulosa cells from diethylstilbestrol-treated immature rats were cultured in a defined medium on collagen-coated plates. Thymidine incorporation was significantly increased by insulin (ED50, 656 +/- 110 ng/ml) and insulin-like growth factor (IGF-I; ED50, 95 +/- 10 ng/ml). Insulin and IGF-I stimulations were amplified by methylisobutylxanthine an inhibitor of phosphodiesterase activity. The effect of both peptides were also enhanced by low doses of (Bu)2cAMP (0.2-1 mM). In contrast, higher concentrations were inhibitory. Similarly, FSH produced a biphasic enhancement of the insulin- and IGF-I-stimulated DNA synthesis. Maximal effects (2- to 6-fold increases) were observed with the lower doses (2-20 ng/ml) of the gonadotropin. FSH enhancement of IGF-I-stimulated DNA synthesis was dependent on cell density. Plating densities of 3-5 x 10(5) cells/cm2 were required for a maximal interaction. It is concluded that FSH, acting through a cAMP-mediated pathway, may regulate granulosa cell proliferation by modulating the mitogenic effects of insulin and/or IGF-I.


Insulin-like growth factor-II and its binding proteins in placental development.

To identify potential mediators or modulators of insulin-like growth factor-II (IGF-II) action in the placenta, we used in situ hybridization to map patterns of gene expression for IGF-II, the functionally related IGF-binding proteins (IGFBPs) 1-4, and the type 1 and 2 IGF receptors in developing rat and term human placentas. IGF-II mRNA was highly abundant in trophoblast-derived elements of the rat placenta from implantation to maturity, except for a significant local reduction in IGF-II gene expression in the junctional zone just before term. IGFBP2 mRNA was barely detected during early placental development, but increased significantly toward term and was most abundant in the junctional zone. The basal plate of the term human placenta showed a similar pattern, with a superficial layer of cytotrophoblasts containing IGF-II mRNA anatomically apposed to a deeper layer of cells expressing IGFBP2 mRNA. Placental IGFBP1, -3, and -4 mRNAs were much less abundant than IGFBP2 and were restricted to the yolk sac and vasculature. Type 1 and 2 IGF receptor mRNAs were abundant and shared the same distribution, together with IGF-II, in the labyrinthine zone. These findings suggest that IGFBP2 may be an important modulator of IGF-II action in placental development. Furthermore, the colocalization of both types of IGF receptor mRNA supports the view that these receptors may compete for IGF-II binding in the placenta.


Phosphorylation of the rodent negative acute-phase protein alpha 1-inhibitor-III by the insulin receptor tyrosine kinase.

alpha 1-Inhibitor-III (alpha 1I3), a broad range proteinase inhibitor, member of the alpha-macroglobulin family, is abundant in normal rat plasma. Insulin-dependent tyrosine phosphorylation of a monomeric 195K glycoprotein (pp195) was observed in wheatgerm agglutinin (WGA)-Sepharose-purified insulin receptor preparations from rat liver and muscle. Phosphorylation of pp195 in vitro required a basic poly-amino acid, i.e. poly-L-lysine. We present evidence identifying pp195 as alpha 1I3. In situ perfusion with saline essentially removed pp195 from rat livers. Addition of normal rat plasma to liver homogenates or to WGA eluates restored insulin-stimulated phosphorylation of pp195; plasma from streptozotocin-diabetic rats was much less effective. Liver-derived pp195 copurified with an abundant plasma protein, with the characteristics of alpha 1I3, on size exclusion and ion-exchange chromatography. An approximately 195K protein, comigrating with alpha 1I3, was markedly diminished in plasma from diabetic rats, and alpha 1I3 concentration was decreased by approximately 70% upon immunoblot analysis. Highly purified alpha 1I3 was phosphorylated by muscle- or liver-derived insulin receptors in the presence of 1 microM poly-L-lysine and comigrated with pp195 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 1I3 phosphorylation was half-maximal at approximately 70 nM and was stimulated by insulin 7-fold. Hindlimb perfusion removed more than 90% plasma albumin but only approximately 20% pp195 from muscles. alpha 1I3 messenger RNA was identified in liver but not in muscle. A specific antibody against alpha 1I3 immunoprecipitated phosphorylated pp195 in WGA-purified insulin receptor preparations from nonperfused liver and from saline perfused and nonperfused muscle. alpha 1I3 is bound and internalized by alpha-macroglobulin receptors; whether it is phosphorylated in vivo is unknown. Hepatic alpha 1I3 synthesis may diminish in diabetic rats.


Alpha 2-macroglobulin expression in the mesometrial decidua and its regulation by decidual luteotropin and prolactin.

During decidualization, cells of the endometrium grow and differentiate giving rise to two different decidual tissues located in either the antimesometrial or mesometrial site of the uterus in the rat. These tissues have different functions in pregnancy. The antimesometrial decidua is an endocrine gland that secretes hormones, whereas the mesometrial decidua appears to play an important role in limiting trophoblast invasion. Since the decidual tissue of the rat produces alpha 2-macroglobulin (alpha 2MG), we examined whether this potent protease inhibitor is specifically expressed by the mesometrial tissue, the site of trophoblast invasion, and whether the alph 2MG gene is regulated by decidual luteotropin (DLt), the PRL-like hormone secreted by the neighboring antimesometrial cells. To determine the secretory proteins of the rat decidua, antimesometrial and mesometrial tissues were dissected out from pseudopregnant rats and cultured with [35S]methionine. The major protein secreted by the antimesometrial cell was the 29-kilodalton decidual luteotropin, whereas a 180-kilodalton protein was predominantly secreted by the mesometrial tissue. Immunoprecipitation studies of 35S-radiolabeled proteins revealed that this high mol wt protein is alpha 2MG and that it is secreted exclusively by the cells forming the mesometrial tissue. To examine whether the alpha 2MG gene was also expressed specifically in the mesometrial decidua, total RNA was isolated from both mesometrial and antimesometrial tissues of day 9-12 pseudopregnant rats and hybridized with alpha 2MG cDNA. Northern blot analysis revealed a 5.4-kilobase message, which was abundantly expressed in the mesometrial decidua. Little, if any, alpha 2MG mRNA was detected in antimesometrial decidua. The ontogeny of the message in the decidua correlated well with the development of the mesometrial tissue. To examine whether alpha 2MG expression is regulated by DLt and/or PRL, a highly specific polyclonal antibody to DLt was generated and decidual tissues were cultured in the presence or absence of DLt antibodies with or without PRL. Neutralization of DLt caused a marked decrease in alpha 2MG mRNA levels. This down-regulation was totally reversed by the addition of PRL and was not affected by alpha 2MG antibodies. In summary, the results of this investigation revealed a compartmentalized gene expression, synthesis and secretion of alpha 2MG in the decidua. The secretion of this protease inhibitor, specifically by the mesometrial tissue which is the site of trophoblast invasion, may be the reason for the minimal amount of tissue damage that occurs during placentation.(ABSTRACT TRUNCATED AT 400 WORDS)


Cytoplasmic estrogen receptors in rat brain: immunocytochemical evidence using three antibodies with distinct epitopes.

The existence of cytoplasmic estrogen receptors (ERs) has been reported in the guinea pig brain using immunocytochemical techniques. While cytoplasmic ERs have been reported recently in other species, such as opossum, musk shrew, and ferrets, an exclusively cell nuclear pattern of ER immunoreactivity has been reported in the rat brain. Because all studies that have reported the existence of cytoplasmic ERs in the brain have used the H 222 monoclonal antibody, the possibility exists that this observation is idiosyncratic to this antibody. In the present experiment three antibodies directed against diverse epitopes on the ER protein were used to immunocytochemically stain ERs in rat brain. With each antibody, ER immunoreactivity was observed in the hypothalamus, preoptic area, amygdala, and midbrain periaqueductal gray. In all cases we observed the highest density of reaction product in cell nuclei, but extensive cytoplasmic immunostaining was observed in most areas as well. In addition to demonstrating the existence of neural ER immunoreactivity in perikaryal cytoplasm and cytoplasmic processes in the brain, this study suggests that the neural cytoplasmic ER immunoreactivity is not just a small fragment of the receptor protein; rather, it is likely to be the entire receptor.


Secretion of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 by intestinal epithelial (IEC-6) cells: implications for autocrine growth regulation.

To identify the factors regulating the proliferation of intestinal epithelium, we examined the effects of various growth factors on [3H] thymidine incorporation into the DNA of IEC-6 cells, an intestinal epithelial cell line derived from rat jejunal crypts. Insulin-like growth factor-I (IGF-I), IGF-II, and insulin stimulated the DNA and protein synthesis of IEC-6 cells in serum-free medium supplemented with transferrin, dexamethasone, and BSA (basal medium). Concentration-response experiments demonstrated that IGF-I is approximately 10 times more potent than IGF-II or insulin in producing 2- to 3-fold stimulations of DNA and protein synthesis by IEC-6 cells. In addition, IEC-6 cells proliferated slowly in the basal medium without any added growth factors. Analysis of medium conditioned by IEC-6 cells by gel filtration chromatography, RIA, HPLC, and N-terminal sequencing revealed that IEC-6 cells synthesize and secrete mature, 7,500 mo wt (M(r)) IGF-II as well as high M(r) forms of IGF-II. In addition, ligand blot, immunoblot, and N-terminal sequence analyses showed that IEC-6 cells produce the 34,000 M(r) IGF-binding protein-2 (IGFBP-2). To determine if IGFBP-2 modulates IGF responses in IEC-6 cells, the IGF-I analogs, Des-(1-3)-IGF-I and [Gln3,Ala4,Tyr15,Leu16]IGF-I, both of which have a reduced affinity for IGFBPs, were tested for their effects on IEC-6 cell proliferation. Both analogs exhibited 10-fold greater potency than IGF-I, presumably because endogenously secreted IGFBPs depress IGF-I binding to cell surface receptors. Finally, purified IGFBP-2 attenuated the DNA synthesis of IEC-6 cells in a dose-dependent manner. We conclude that IGFBP-2 secreted by intestinal epithelial cells is capable of limiting the mitogenic activity of both exogenous and endogenous IGFs by blocking the association of the growth factors with cell surface binding sites. These results further suggest that the growth of IEC-6 cells is modulated by autocrine mechanisms involving IGF-II and IGFBP-2.


Arginine vasopressin stimulates atrial natriuretic peptide gene expression and secretion from rat diencephalic neurons.

The central nervous system modulates cardiovascular function and fluid and electrolyte balance in part through the actions of vasoactive peptides/neurotransmitters. The presence of several vasoactive peptides and their receptors in the hypothalamus suggests a possible interaction at this site. One level at which vasoactive peptides such as arginine vasopressin (AVP) and atrial natriuretic peptide (ANP) might interact is through the mutual regulation of production and secretion in the hypothalamus. To determine whether AVP modulates ANP gene expression and secretion, we cultured fetal rat diencephalic neurons in the presence of AVP. AVP induced a significant increase in ANP secretion in dose-related fashion (mean +/- SEM basal ANP, 87 +/- 4 pg/ml; maximal mean AVP-stimulated ANP, 146 +/- 6 pg/ml; P less than 0.05, by analysis of variance). Neither oxytocin nor the vasoactive neuropeptide angiotensin-II had any effect on ANP secretion. The stimulatory effect of AVP was significantly blocked by coincubation with a V1 receptor antagonist, but was unaffected by a V2 receptor antagonist. The immunoreactive ANP secreted in response to AVP was the major brain isoform, ANP-(103-126). Coincubation with a calcium channel antagonist, nifedipine, had no effect on AVP-induced ANP secretion, while ryanodine, an inhibitor of intracellular calcium mobilization, significantly reduced the stimulatory effect of AVP. AVP induced a dose-related, nearly 3-fold maximal increase in ANP mRNA expression at 4 h. Coincubation of the neurons with a V1 receptor antagonist also significantly attenuated the increased ANP gene expression induced by AVP. These results indicate that AVP acts directly through V1 receptors on cultured fetal rat diencephalic neurons to augment ANP gene expression and secretion of the peptide. The effects are probably related to AVP-stimulated mobilization of intracellular calcium and not the result of calcium influx into the cell. These studies provide the first evidence that AVP modulates ANP production from cultured neurons. In the central nervous system, these two vasoactive neuropeptides might interact in part through the regulation of ANP production by AVP.


Insulin-like growth factor binding protein expression in the hypothyroid rat is age dependent.

Thyroid hormone is essential for normal growth and development. For certain T4 effects, there is a critical period during ontogeny when normal T4 levels are required, and thyroid replacement after that period cannot correct the changes in hypothyroid animals. We have previously described a prolonged high expression of serum insulin-like growth factor binding protein (IGFBP)-2 during the perinatal period in congenitally hypothyroid rats. To see if this effect was confined only to a certain period during rat ontogeny, we made rats hypothyroid with methimazole treatment either prenatally, or at different postnatal ages from 1 to 14 days of life, and at adult age. Serum IGF-I levels were reduced by approximately 30% in all the 18-day-old hypothyroid animals, and did not correlate with the duration of the hypothyroid state. Serum IGF-I levels in the adult animals were 50% of control levels. At the age of 18 days, control animals had only very low levels of IGFBP-2 demonstrable by western ligand blotting, whereas the congenitally hypothyroid animals had elevated levels. Pups placed on methimazole treatment since the first day of life showed higher IGFBP-2 levels at the age of 18 days, although the change was not as prominent as in the congenitally hypothyroid animals (200% vs. 500% of control levels, respectively). Binding protein changes were approximately 2-fold at the mRNA level. Rats started on methimazole after the first 5 days of life showed normal low levels of IGFBP-2 at the age of 18 days. Abnormal IGBFP-2 expression in congenitally or neonatally hypothyroid animals could be corrected by thyroid hormone replacement, if started during the first week of the life, but not later. In adult hypothyroid animals, there was no induction of IGFBP-2 expression, but the levels of IGFBP-3 and -4 were decreased to 80% and to 30% of control levels, respectively. IGFBP-3 messenger RNA (mRNA) levels were decreased to 50% of control levels but IGFBP-4 mRNA levels were paradoxically increased in the hypothyroid animals. All these changes could be corrected by T4 replacement. In conclusion, there exists a critical period during the perinatal development of the rat, when thyroid hormone is essential for a subsequent normal IGFBP-2 ontogenic pattern. Adult animals show a completely different IGFBP response to hypothyroidism, with a decrease of IGFBP-3 and -4 levels. Thus, the effects of thyroid hormone on IGF-IGFBP axis regulation depend on the developmental stage of the animal.


Spatio-temporal expression of estrogen sulfotransferase within the hepatic lobule of male rats: implication of in situ estrogen inactivation in androgen action.

Estrogen sulfotransferase (EST) catalyzes transfer of the sulfate group from phosphoadenosine phosphosulfate to estrogenic steroids. Since estrogen sulfates do not bind to the estrogen receptor with high affinity, EST can control the intracellular level of the receptor-active estrogens. Androgen action in the rat liver, as indicated by the androgenic induction of alpha 2u-globulin, is inhibited by low levels of estrogens. Thus, in situ estrogen inactivation by EST is expected to increase hepatic androgen sensitivity. During the lifespan of the animal, rat liver undergoes three distinct phases of androgen sensitivity, i.e. prepubertal androgen insensitivity, androgen sensitivity after approximately 40 days of age, and androgen insensitivity during senescence (greater than 750 days). EST in the liver is expressed only after puberty, when the liver becomes androgen sensitive. Furthermore, localization of EST and its corresponding mRNA within the lobular unit of the liver demonstrates that only androgen-responsive hepatocytes located around the central vein contain immunoreactive EST and its corresponding mRNA. These temporal and spatial correlations of EST expression and hepatic androgen sensitivity support the concept that steroid-inactivating enzymes play important roles in sex hormone action.


Inhibition of human fibroblast insulin-like growth factors binding protein (IGFBP) production by IGFBP-3.

Human neonatal fibroblasts in monolayer culture secrete a number of insulin-like growth factor binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [125I]IGF-II ligand blotting, human neonatal fibroblasts secrete IGFBP-3, an IGFBP of 29-31 kDa, and a 22-24 kDa IGFBP after treatment with 50 ng/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-1000 ng/ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 ng/ml) IGF-I, IGFBP-3 had approximately 20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming growth factor-beta 1 (TGF beta 1), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGF beta 1 had 2 to 3-fold reduced potency. These results demonstrate that fibroblast IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between fibroblast IGFBPs.


Implantation of self-expanding esophageal metal stents for palliation of malignant dysphagia.

Eleven self-expanding metal stents were perorally implanted in ten patients with locally advanced malignant obstruction of the esophagus. After bougienage of the strictures, the stents were painlessly inserted and properly released by means of an 18 French gauge delivery catheter. In all cases, the endoprostheses expanded to a diameter of 14-20 mm and achieved immediate improvement of dysphagia. One perforation was seen after a single session of dilatation and subsequent stent insertion. No other early complication was observed. After a median follow-up of 74 days (Range, 33-252 days), one of eight patients is still alive and 7 died of non-procedural causes. The grade of dysphagia improved from a mean of 2.9 to a mean of 1.6 and 2.0, respectively, depending on the follow-up period (scale 0-4). Esophageal reobstruction occurred in four patients due to food impaction (two patients) or tumor ingrowth into the stent through the wire mesh (two patients). Recanalisation of the obstructed stent lumen was achieved by endoscopic irrigation (two patients), laser therapy only (one patient) or diathermia with subsequent insertion of a conventional plastic endoprosthesis into the metal stent (one patient). The initial results are promising. The delivery system, the wide-bore diameter, the macroporous configuration and the low mass of the self-expanding stents would seem to be associated with a less traumatic insertion procedure and a lower rate of stent migration as compared with conventional prostheses. Technical improvement may be required for prevention of tumor infiltration. Controlled trials are warranted to determine the future role of metallic stents for palliation of esophagocardial tumors.


Self-expanding metal stents for palliation of malignant esophageal obstruction--a pilot study of eight patients.

We sought to determine whether the application of a self-expanding metal stent enables palliation of malignant dysphagia with minimal risk. The results of pilot studies from two centers are reported. We treated 8 inoperable patients with a 14 mm self-expanding metal stent (Wallstent). The stent was applied without general anesthesia under mild i.v. sedation. The procedure was successful in all cases. No side effects were noted. In one patient, tumor ingrowth through the meshes of the stent occurred. This patient was additionally treated with a percutaneous gastrostomy. One patient experienced tumor overgrowth of the proximal end, necessitating laser treatment. Three patients were still alive after three months. The mean number of cumulative endoscopic interventions per patient was 2.2 (SD: +/- 2; median 2). The mean observation time was 10.7 weeks +/- 2 (median 12). Dysphagia was graded from 0 (normal swallowing) to 4 (inability to swallow saliva). Dysphagia was significantly (p less than 0.0005) reduced from grade 3.1 (SD: +/- 0.35) to 0.5 (SD: +/- 0.5) immediately after stenting. 62.5% of the patients were able to manage a virtually normal diet (in one of these patients dysphagia recurred six weeks after stent placement due to tumor ingrowth). Six patients (75%) were able to ingest all necessary calories orally. The application of a 14 mm self-expanding metal stent in cases of inoperable malignant esophageal obstruction seems to offer safe and effective palliation of malignant dysphagia.


Self-expanding metal stents for palliation of malignant gastric outlet obstruction.

Treatment of inoperable gastric outlet obstruction caused by tumor compression is difficult and often unsatisfactory. The case of a 56-year-old female patient in whom self-expanding stents were implanted, successfully enabling oral nutrition, is reported.


Analysis of human tear proteins by two-dimensional electrophoresis.

Human tear proteins in the conjunctival sac were separated on the basis of the differences in their isoelectric points and molecular weights using micro two-dimensional electrophoresis combined with immunoblotting. The two-dimensional electrophoretic patterns of tear proteins from patients with conjunctivitis were compared with those from normal individuals. We also measured integrated intensities of seven protein spots, lactoferrin (LF), albumin and five specific tear proteins (STP), to examine differences in the amounts of these proteins in tears from normal individuals of different sexes. In the tears from patients with conjunctivitis, secretory immunoglobulin A (IgA), LF and STP spots were stained more weakly, whereas the albumin spot was stained more strongly as compared with those from normal individuals. Furthermore, haptoglobin and IgG spots appeared in the tears from patients with conjunctivitis. These were more prominent in the tears from patients with severe conjunctivitis. There were significant differences in the amounts of LF and two kinds of STPs in the different sexes. The amounts of these proteins were larger in females.


Specific polypeptide markers in chronic B cell malignancies detected by two-dimensional gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of silver stained polypeptides was used to discriminate the cell protein profiles in chronic B cell malignancies. We identified 5 distinct polypeptides (H1 to H5) present in hairy cell leukemia (HCL) (14 cases) and absent in most chronic lymphocytic leukemia (CLL) and normal B lymphocytes. Two polypeptides, H2 and H5, were absolutely specific of HCL. The 2-D PAGE profiles found in 3 splenic lymphoma with villous lymphocytes (SLVL) and 2 HCL variants (HCL-V) were similar to HCL with a unique additional peptide present in 3/3 SLVL and 1/2 HCL-V. When HCL and SLVL were assessed for the CLL stage-specific 2-D PAGE patterns, HCL was related to stage A/B while SLVL was related to stage C. In conclusion, 2-D PAGE analysis of the molecular pattern of B lymphocytes provides a new means to recognize the leukemic origin of the sample and distinguish HCL from CLL and SLVL.


Molecular architecture of acetylcholinesterase collagen-tailed forms; construction of a glycolipid-tailed tetramer.

Asymmetric forms of Torpedo acetylcholinesterase (AChE) are produced in COS cells by the simultaneous expression of collagenic subunits (Q) and catalytic T subunits (AChET). Truncated AChET delta subunits, from which most of the C-terminal peptide (TC) had been deleted by mutagenesis, did not associate with Q subunits. The TC peptide is therefore necessary for the association of the AChET and Q subunits. In order to determine the orientation of the Q subunit in the collagen-tailed forms, we have developed an antiserum against its non-collagenic C-terminal domain, expressed as a fusion protein in Escherichia coli. This antiserum, which recognized the Q subunit in Western blots, was found to react with intact asymmetric forms, but not with collagenase-treated forms, from which the distal part of the tail had been cleaved, suggesting that the N-terminal non-collogenic domain (QN) is responsible for the interaction with the AChET subunits. This was confirmed by creating a chimeric subunit (QN/HC), in which QN was linked to the C-terminal peptide of the H subunit of Torpedo AChE, which contains the glycophosphatidylinositol (GPI) cleavage/attachment signal: co-expression of AChET and QN/NC produced GPI-anchored tetramers, which were sensitive to PI-PLC and largely exposed to the external surface of the cells. We thus demonstrate that: (i) the HC peptide is sufficient to determine the addition of a glycolipid anchor and (ii) the QN domain is sufficient to bind a catalytic AChET tetramer by interacting with the TC peptide.


Interaction of the RNA-binding domain of the hnRNP C proteins with RNA.

The hnRNP C proteins are among the most abundant and avid pre-mRNA-binding proteins and they contain a consensus sequence RNA-binding domain (RBD) that is found in a large number of RNA-binding proteins. The interaction of the RBD of the hnRNP C proteins with an RNA oligonucleotide [r(U)8] was monitored by nuclear magnetic resonance (NMR). 15N and 13C/15N-labelled hnRNP C protein RBD was mixed with r(U)8 and one- and two-dimensional (1D and 2D) NMR spectra were recorded in a titration experiment. NMR studies of the uncomplexed 93 amino acid hnRNP C RBD (Wittekind et al., 1992) have shown that it has a compact folded structure (beta alpha beta beta alpha beta), which is typical for the RBD of this family of proteins and which is comprised of a four-stranded antiparallel beta-sheet, two alpha-helices and relatively unstructured amino- and carboxy-terminal regions. Sequential assignments of the polypeptide main-chain atoms of the hnRNP C RBD-r(U)8 complex revealed that these typical structural features are maintained in the complex, but significant perturbations of the chemical shifts of amide group atoms occur in a large number of residues. Most of these residues are in the beta-sheet region and especially in the terminal regions of the RBD. In contrast; chemical shifts of the residues of the well conserved alpha-helices, with the exception of Lys30, are not significantly perturbed. These observations localize the candidate residues of the RBD that are involved in the interaction with the RNA.(ABSTRACT TRUNCATED AT 250 WORDS)


Monoclonal antibodies to NF-Y define its function in MHC class II and albumin gene transcription.

NF-Y is a sequence-specific DNA-binding protein which, as a heterodimer, recognizes CCAAT motifs in a variety of transcriptional promoters. We have generated a panel of monoclonal and affinity-purified polyclonal antibodies directed against various epitopes of NF-Y. These reagents are highly specific for either of the A or B subunits; we have mapped the epitopes recognized by the monoclonal antibodies to the glutamine-rich activation domain of NF-YA. The antibodies inhibit in vitro transcription from the promoters of the albumin gene and of Ea, a class II gene of the major histocompatibility complex. These data definitively demonstrate the role of NF-Y in regulating the transcription of two tissue-specific genes whose expression patterns do not overlap. Interestingly, the antibodies cannot inhibit a formed pre-initiation complex, but do block reinitiation of subsequent rounds of transcription from the same templates.


Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome.

Hepadnaviruses, as well as other pararetroviruses, express their pol (P) gene product unfused to the preceding core gene implying that these retroelements have developed a mechanism for initiating assembly and replication that is principally different from the one used by retroviruses and retrotransposons. We have analysed this mechanism for the human hepatitis B virus by using a newly developed, highly sensitive detection method based upon radiolabelling of the P protein at newly introduced target sites for protein kinase A. The results obtained demonstrate that polymerase encapsidation depends on the concomittant encapsidation of the HBV RNA pregenome and that packaging of the viral RNA, in turn, depends on the presence of P protein. Loss of P protein encapsidation by mutations inactivating the HBV RNA encapsidation signal epsilon could be compensated by trans-complementation with recombinant RNA molecules carrying the epsilon sequence. Thus, in contrast to retroviral replication, the interaction of the hepadnaviral P protein and the RNA genome at its packaging signal appears to be crucial for initiating the formation of replication-competent nucleocapsids. Furthermore, RNA control of P protein packaging stringently limits the number of polymerase molecules that can be encapsidated.


Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme.

The DNA binding activity of Fos and Jun is regulated in vitro by a post-translational mechanism involving reduction-oxidation. Redox regulation occurs through a conserved cysteine residue located in the DNA binding domain of Fos and Jun. Reduction of this residue by chemical reducing agents or by a ubiquitous nuclear redox factor (Ref-1) recently purified from Hela cells, stimulates AP-1 DNA binding activity in vitro, whereas oxidation or chemical modification of the cysteine has an inhibitory effect on DNA binding activity. Here we demonstrate that the protein product of the ref-1 gene stimulates the DNA binding activity of Fos-Jun heterodimers, Jun-Jun homodimers and Hela cell AP-1 proteins as well as that of several other transcription factors including NF-kappa B, Myb and members of the ATF/CREB family. Furthermore, immunodepletion analysis indicates that Ref-1 is the major AP-1 redox activity in Hela nuclear extracts. Interestingly, Ref-1 is a bifunctional protein; it also possesses an apurinic/apyrimidinic (AP) endonuclease DNA repair activity. However, the redox and DNA repair activities of Ref-1 can, in part, be distinguished biochemically. This study suggests a novel link between transcription factor regulation, oxidative signalling and DNA repair processes in higher eukaryotes.


Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation.

IRS-1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3'-kinase, but p85 is not tyrosyl phosphorylated. IRS-1 contains nine tyrosine phosphorylation sites in YXXM (Tyr-Xxx-Xxx-Met) motifs. Formation of the IRS-1-PtdIns 3'-kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3'-kinase to IRS-1 is mediated through the SH2 (src homology-2) domains of p85. Furthermore, overexpression of IRS-1 potentiates the activation of PtdIns 3-kinase in insulin-stimulated cells, and tyrosyl phosphorylated IRS-1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3'-kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS-1 to the SH2 domains of p85 is the critical step that activates PtdIns 3'-kinase during insulin stimulation.


Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.

The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.


Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.


Growth hormone treatment in children with preterminal chronic renal failure: no adverse effect on glomerular filtration rate.

Impaired growth and stunting remains a major therapeutic problem in children with chronic renal failure (CRF). Recombinant human growth hormone (rhGH) treatment may be beneficial, but concern has been raised about possible side-effects, i.e. deterioration of renal function and glucose intolerance. We have treated 10 prepubertal children with CRF (median age 7.5 [1.7-10.0] years) with 4 IU rhGH/m2 per day s.c. over a period of 1 year. Height velocity increased significantly (P less than 0.03) from basal 4.6 (2.0-14.0) cm/year to 9.7 (6.8-17.6) cm/year. Height velocity SDS for chronological age and for bone age increased in all children from basal median -2.3 to +3.8 (P less than 0.005). Median glomerular filtration rate (GFR) measured by single injection inulin clearance at onset was 18 (11-66) ml/min per 1.73 m2 and did not change significantly during the treatment year. The loss of GFR as estimated by creatinine clearance was similar during the treatment year (median loss 1.3 ml/min per 1.73 m2) compared to the year before treatment (median loss 3.7 ml/min per 1.73 m2). Serum glucose levels during an oral glucose tolerance test did not change, but fasting as well as stimulated insulin levels increased significantly with time during the study period. It is concluded that the rhGH regimen employed was remarkably effective in improving growth velocity in children with CRF without adversely affecting GFR. Glucose homeostasis remained stable, but at the expense of increased serum insulin levels.


Assessment of new immunosuppressive drugs in a rat cardiac allograft heterotopic model.

We assessed FK506 (FK) and rapamycin (RPM) in a heterotopic abdominal rat heart transplant model using a major histocompatibility mismatch (DA to LEW). The end point of our study was histologic grading of rejection (Billingham and working formulation) at 1 week. Two doses of FK (2.0 and 8.0 mg/kg p.o., q.d.) and RPM (1.5 and 6.0 mg/kg i.p., q.d.) were compared to allografts without and with ciclosporin (12.5 mg/kg p.o., q.d.) treatment. Results show: (1) weak heartbeat and full rejection on day 5 in all untreated allografts; (2) weak heartbeat and high degree of rejection in groups receiving low doses of FK and RPM; (3) strong heartbeat and mild rejection in both high FK and RPM dose groups comparable to the results of the hearts treated with ciclosporin; (4) 1 animal in each high FK and RPM dose group showed possible signs of toxicity, and (5) the strength of the heartbeat was not a reliable indicator of the efficacy of an immunosuppressive drug. We conclude that even in a major histocompatibility mismatch model at the time of the strongest immune response (1 week), all three tested drugs can reduce the degree of rejection from severe (untreated allografts) to mild if given in an adequate dosage.