Changes in rapidly transported proteins associated with development of abnormal projections in the diencephalon.

The development of the hamster visual system is accompanied by striking changes in the pattern of proteins that are synthesized in retinal ganglion cells and rapidly transported to their nerve terminals. To determine whether any of these protein changes are regulated by interactions between the developing nerve endings and the cells with which they form synapses, we induced retinofugal axons to form abnormal projections in the lateral posterior (LP) nucleus of the thalamus and dense patches of hyperinnervation in the lateral geniculate nucleus (LGN) by removing their principal target, the superior colliculus (SC), the day after birth. Under these experimental conditions, two rapidly transported proteins, including the neural cell adhesion molecule, NCAM, showed significant changes in their time course of expression. NCAM, identified here using a monospecific antibody, is normally synthesized and transported at high levels at early stages of development and then declines during the second and third postnatal weeks. However, this decline was delayed when optic fibers were re-routed. A second rapidly transported protein, M(r) = 67 kDa, pI = 4.7, normally shows a rise in its synthesis and transport during terminal arbor formation and a subsequent decline, but it also remained elevated for a prolonged period when the SC was absent. These findings cannot be accounted for by a simple delay in the retinal ganglion cells' program of axonal growth, since other rapidly transported proteins, including the growth-associated protein GAP-43, showed a normal developmental time-course when the SC was removed. Target interactions therefore appear to influence the retinal ganglion cells' expression of different proteins in a specific fashion.


Anatomical and physiological study of interneurons in an oligosynaptic cutaneous reflex pathway in the cat hindlimb.

Morphological and physiological studies were undertaken in the cat lumbosacral spinal cord in order to examine individual last-order excitatory interneurons in the disynaptic pathway between low threshold superficial peroneal (SP) afferents and flexor digitorum longus (FDL) motoneurons. Last-order interneurons projecting to the FDL motor nucleus were visualized with activity-dependent trans-synaptic transport of lectin-conjugated horseradish peroxidase (WGA-HRP). The location of lumbar interneurons that responded at short latencies (less than 3 ms) to electrical stimulation of the SP nerve was further defined using extracellular recording. The projection of individual interneurons to motoneurons in the L6 segment was defined by spike-triggered averaging of population ventral root potentials (VRPs) using the sucrose gap technique. Measurements of the central latencies from the arrival of the SP afferent volley to interneuron firing, and from interneuron spike to the onset of the VRP EPSPs, confirm the existence of a low threshold, disynaptic SP pathway that excited motoneurons. These data provide useful latency constraints for the interpretation of the synaptic length of cutaneous reflex pathways in the cat hindlimb.


Neurokinin A-like immunoreactivity in articular afferents of the cat.

Dorsal root ganglion cells with axons innervating the cat's knee joint via the medial articular nerve were retrogradely labelled with Fast blue. Neurokinin A-like immunoreactivity was found in 4.5 +/- 1.1% (mean +/- S.D. of 5 nerves and 695 cells) of the articular afferents. Colchicine treatment of the ganglia increased the percentage of immunopositive cells to 8.5 +/- 0.7% (mean +/- S.D. of 6 nerves and 554 cells) after 3-22 h. The diameter distribution of the immunopositive somata ranged from 20 to 50 microns with a maximum at 26-30 microns. Comparing the proportions of neurokinin A-immunopositive cells with those of substance P, it can be calculated on the basis of mRNA encoding that neurokinin A is synthetized in about half of the substance P-containing primary articular afferents.


Effects of intracerebroventricularly administered neostigmine on hypothalamic monoaminergic neuronal activities in awake rats.

Hypothalamic neuronal activities of noradrenaline, dopamine and serotonin as well as serum glucose concentrations were simultaneously monitored 60 min after the third cerebroventricular injection of neostigmine, a cholinesterase inhibitor, in rats. Each neuronal activity was assessed from a ratio of the concentration of the major metabolite to that of its precursor monoamine itself by using the technique of gas chromatography-mass spectrometry. Neostigmine caused significant increases in serum glucose concentrations, hypothalamic noradrenergic and dopaminergic neuronal activities, and significantly suppressed hypothalamic serotonergic neuronal activity. All these responses to neostigmine were completely inhibited by the co-administration of atropine. These observations emphasize the important role of the interactions between cholinergic (muscarinic) and monoaminergic neurons in the brain.


Cyclic AMP and the nicotinic response of bovine adrenal chromaffin cells.

The effects of cAMP analogs on the nicotinic responses of bovine adrenal chromaffin cells were examined by monitoring nicotine-induced whole cell currents, [3H]norepinephrine release, and membrane conductance changes. None of the three methods revealed an increased nicotinic response after treatment with cAMP analogs. The compounds did increase [3H]norepinephrine release from the cells but the effect was not exerted at the level of nicotinic receptors. Bovine adrenal chromaffin cells differ in this respect from chick ciliary ganglion neurons which do show increased nicotinic responses after treatment with cAMP analogs.


Substance P inhibits carbamylcholine-stimulated 22Na+ efflux from acetylcholine receptor-enriched Torpedo electroplaque membrane vesicles.

The effect of substance P on nicotinic acetylcholine receptor function was examined in Torpedo electroplaque membranes. The peptide inhibited carbamylcholine-stimulated 22Na+ efflux in a concentration-dependent manner. By irreversibly blocking spare receptors with alpha-bungarotoxin, the IC50 for substance P was shown to be less than 3 microM. Inhibition by substance P was slow relative to receptor activation by carbamylcholine, consistent with an enhancement of desensitization or a slow allosteric block.


[Isolation and characterization of monoclonal antibody directed against antigenic peptide presented by class I histocompatibility molecule].

We describe the isolation and several characteristics of a monoclonal antibody (X5.3.7) which recognizes a peptide derived from influenza virus nucleoprotein and presented by the murine class I major histocompatibility molecule Kd. X5.3.7 is thus an example of an antibody capable of recognizing an epitope normally recognized by T cells.


[Neurotransmission and sudden infant death. Study of cerebrospinal fluid].

The study of 33 cerebrospinal fluids of infants victims of sudden death shows a very significant increase of the metabolites of dopamine and serotonin. These determinations, compared to a control group, indicate a failure, concerning these two neurotransmitters, which could induce a cardiorespiratory seizure. This failure has likely a multifactorial origin.


Nicotinic and muscarinic modulation of 5-hydroxytryptamine (5-HT) release from porcine and canine small intestine.

Strips of porcine and canine small intestine were incubated in vitro and the release of 5-hydroxytryptamine (5-HT) was determined by HPLC with electrochemical detection. The spontaneous outflow of 5-HT from the porcine and canine small intestine largely reflects calcium-dependent 5-HT secretion from enterochromaffin cells which are under a spontaneous neuronal, excitatory input as indicated by the inhibitory effect (30-40%) of tetrodotoxin. In both species, nicotine enhanced the release of 5-HT in a concentration-dependent manner by a maximum of about 50% at 100 microM. This effect was blocked by the nicotine receptor antagonist hexamethonium, but not by the subtype-selective nicotine receptor antagonist alpha-bungarotoxin. The effect of nicotine was rapidly desensitized. The presence of tetrodotoxin abolished the effect of nicotine on 5-HT release in canine tissue but not in porcine tissue. The presence of the muscarine receptor antagonist scopolamine prevented the effect of nicotine on 5-HT release from canine tissue, but significantly enhanced 5-HT release from porcine tissue. The muscarine receptor agonist oxotremorine inhibited 5-HT release from porcine tissue, but increased 5-HT release from canine tissue. However, in the presence of tetrodotoxin, oxotremorine enhanced 5-HT release in tissue from both species. In conclusion, activation of nicotine receptors induce the release of 5-HT from porcine and canine small intestine. In the dog, the effect of nicotine is mediated via the release of acetylcholine which then stimulates 5-HT release via muscarine receptors on the enterochromaffin cells. In the pig, the stimulatory effect of nicotine appears to be located directly on the enterochromaffin cells. In addition, activation of neuronal muscarine receptors in the porcine small intestine induced the release of a previously unidentified neurotransmitter which inhibited 5-HT release. Nicotine, via cholinergic interneurons, also appears to induce the release of this inhibitory neurotransmitter which opposes the direct stimulatory action of nicotine on 5-HT release.


[Potentiation of the antitumoral effect of electrochemotherapy by immunotherapy with allogeneic cells producing interleukin 2].

Electrochemotherapy (ECT) is a new antitumour treatment which consists of delivering electric pulses to the tumour a few minutes after an intravenous injection of bleomycin. The antitumour efficacy of ECT is increased by local injections of interleukin 2 (IL2) in the oedema which appears at the site of the treated tumours. We have shown that tumour cells inoculated in syngeneic mice are rejected if IL2 secreting allo- or xenogeneic cells are co-injected with tumour cells. We report here the large increase of ECT therapeutical efficacy when allogeneic cells secreting IL2 are injected into the peri-tumoural oedema.


Channels activated by stretch in neurons of a helix snail.

Single-channel recordings from central neurons of the helix snail, Cepaea nemoralis, revealed two types of channels that could be activated by stretch (i.e., by the membrane deformation produced when suction is applied to the patch pipette). One, a K+ channel (58 pS in physiological solution), was evident in excised and cell-attached patches. Its conductance in symmetrical [K+] solutions indicated a channel of high K+ permeability (PK = 3.4 x 10(-13) cm/s). Though osmoregulation has been suggested as a function for such channels, comparisons among molluscs indicate osmotic milieu does not govern their expression; Cepaea is terrestrial, and stretch-activated K+ channels similar to those described here occur in aquatic and marine molluscs. The second type of channel, observed only in excised patches, was Cl- permeant; it had a large conductance (130 pS) and was inactive prior to patch excision. Membrane tension may not be the physiological activator of either the K+ or Cl- channel; the channels are designated as stretch-activated channels on the basis of their experimental behaviour during single-channel recording.


Partial inhibition of skeletal muscle contraction by dantrolene sodium and its modification with perchlorate and Bay K 8644.

The effects of dantrolene sodium (DAN) on the dihydropyridine receptor (DHPR) of the transverse (T) tubule voltage sensor (Ca2+ channel) was studied with single fibers from bullfrog toe muscle. Perchlorate (ClO4-), which acts selectively on the DHPR, overcame DAN-induced inhibition of twitch tension. Bay K 8644, a DHPR agonist, slowed the rate of twitch inhibition by DAN. DAN inhibited twitch tension to a greater extent in Ca(2+)-free solution than in Ringer solution or solution containing Zn2+, whereas twitch inhibition by DAN was less in caffeine-containing solution than in the control. The effects of DAN on Zn(2+)- and caffeine-treated fibers and on fibers in Ca(2+)-free solution suggest that DAN must act near the voltage sensor of the T tubule. However, differences in net twitch inhibition by DAN between control fibers and fibers potentiated by ClO4- or Bay K 8644 suggest that DAN does not bind to the same site as these potentiating agents do. The role of myoplasmic Ca2+ in DAN-induced inhibition of twitch and the effects of DAN on the mechanical threshold and membrane potential in skeletal muscle are discussed.


Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.


Mode of antibacterial action of dodine (dodecylguanidine monoacetate) in Pseudomonas syringae.

Treatment of Pseudomonas syringae cells with 50 microM dodecylguanidine monoacetate (dodine) resulted in the rapid degradation and release of RNA and cell lysis. Higher concentrations resulted in a progressive decrease in the intensity of these responses, and the appearance of extensive zones of coagulated cytoplasm, indicating that the decrease in RNA degradation probably resulted from an inhibition of the RNases, due to protein denaturation. Dodine also induced expansion of the outer membrane, with the formation of protuberances and intracellular myelin-like structures, which were already evident after 1 min of treatment, indicating that dodine is able to cross the outer and cytoplasmic membranes rather rapidly, and to form, alone or in combination with cell phospholipids and proteins, considerable amounts of triple-layered profiles. In P. syringae cells, saturation levels of dodine corresponded to more than five times the amount needed to form a close-packed monolayer of dodine on the cell surface. The different membranous structures formed in dodine-treated cells, and the coagulation of the cytoplasm, seem to be responsible for the uptake of such high amounts of dodine. The uptake isotherm was essentially Langmuirian. The results presented in this and previous reports indicate that the antibacterial activity of dodine on P. syringae is mainly the result of the action of micelles of the surfactant.


Substance-P-like immunoreactive amacrine cells in the adult and the developing rat retina.

Substance-P-like immunoreactivity (SP-LI) cells in the Long-Evans rat retina were investigated by combining immunohistochemistry with [3H]thymidine autoradiography. Two subpopulations of SP-LI amacrine cells, with cell bodies in either the proximal portion of the inner nuclear layer (INL) or the ganglion cell layer (GCL), were identified based on morphology, pattern of distribution and development. In the INL, SP-LI cells were found scattered throughout the retina. However, in the GCL, they were limited to the superio-temporal region. Such a contrast in distribution specific to nuclear layers was present upon first detection of SP-LI amacrine cells and persisted throughout development. Birthdating revealed a temporal lag in the histogenesis of SP-LI cells situated in the GCL relative to that in the INL, suggesting that the two subpopulations developed separately. Overall, unique anatomical features of the SP-LI amacrine cells in the rat retina were observed which could only have been uncovered through detailed analyses in the adult as well as during postnatal development.


alpha-fetoprotein and screening markers of congenital disease.

Alpha-fetoprotein (AFP) is produced in the gut and liver during fetal life and appears to act like albumin in the adult. Because AFP appears in the maternal circulation during pregnancy, interest has been focused on its measurement in maternal serum to predict fetal abnormality. In addition, AFP, as an embryonic product, is elevated in certain malignant states. This article provides a summary of current clinical knowledge of AFP and its applications.


Serum concentrations of immune parameters during acute cardiogenic pulmonary edema.

To evaluate the effect of acute cardiogenic pulmonary edema on the concentrations of immune parameters in serum. Prospective, controlled study. Medical ICU. Twenty-four consecutive patients with acute pulmonary edema who had significant clinical improvement within 30 mins and did not show any evidence of either tissue damage or infection. For comparison, 25 healthy, age-matched controls and 25 patients with mild chronic heart failure were also studied. Treatment with oxygen, nitrates, and loop diuretics. Lymphokines, acute-phase reactants, and cortisol concentrations were measured in serial serum and plasma samples. Serum concentrations of soluble CD-8 antigen (soluble CD-8) decreased from 928 +/- 124 (SEM) U/mL on admission to 712 +/- 112 and 579 +/- 67 U/mL after 2 and 6 hrs, respectively (p less than .05, p less than .01), and returned to baseline values within 48 hrs (853 +/- 109 U/mL). Concentrations of soluble interleukin-2 receptor increased from 721 +/- 71 to 1078 +/- 112 and 1226 +/- 128 U/mL 12 and 36 hrs, respectively, after admission (p less than .05, p less than .01). Plasma cortisol concentrations were markedly increased on admission (56.9 +/- 4.7 vs. 13.1 +/- 1.3 micrograms/dL after recovery, p less than .001). Increased cortisol concentrations coincided with the nadir of soluble CD-8. Tumor necrosis factor-alpha remained within normal limits in all patients. Neither acute-phase reactants nor angiotensin converting enzyme activity showed significant changes during the observation period. The present results indicate significant alterations in the serum concentrations of immune parameters as an effect of an uncomplicated acute cardiogenic pulmonary edema.


Effect of bicarbonate on intracellular potential of rabbit ciliary epithelium.

Extracellular HCO3- hyperpolarizes the intracellular potential and makes the aqueous medium negative with respect to the stromal surface of the rabbit ciliary epithelial syncytium. The bases for these observations have been unclear. We have been studying the bicarbonate-induced hyperpolarization (BIH) with sustained intracellular recordings for periods as long as 1-2 hrs. The BIH was observed [6.0 +/- 0.4 mV (mean +/- SE, N = 22)] even when the external pH was clamped constant by appropriately changing the CO2 tension. External HCO3- was required since aeration with CO2 at low external pH did not replicate the BIH. DIDS [4,4'-diisothiocyano-2,2'-disulfonic acid] did not abolish the effect. The hyperpolarization is unlikely to reflect the pH dependence of K+ channels alone, since the effect was not reduced by either 2 mM Ba2+ alone or 2 mM Ba2+ together with 50-100 microM quinidine. The BIH depends directly or indirectly on external Na+, since the sign of the polarization response was reversed either by replacing Na+ with N-methyl-D-glucamine or by blocking the Na+,K(+)-exchange pump with 50-100 microM ouabain. Replacement of external Cl- with NO3- or application of the Cl(-)channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate] depolarized the membrane and reversed the sign of the BIH. The response of the ciliary epithelium to HCO3- is complex and may arise from several mechanisms. We suggest that one important element is an anion channel whose conductance is reduced by bicarbonate and whose reversal potential is indirectly dependent on the operations of the Na+,K(+)-pump and a Cl(-)-linked symport.


Dependence of presaccadic cortical potentials on the type of saccadic eye movement.

Premovement cortical potentials were studied with 4 types of saccadic eye movement: (a) visually triggered saccades of normal reaction time (RT; regular saccades); (b) visually triggered saccades of extremely short RT (express saccades); (c) saccades towards predicted target locations (anticipatory saccades); (d) saccades back towards predicted location of fixation point (refixation saccades). With all 4 saccade types a "presaccadic negativity" with the maximum at the vertex (Cz) was observed. A bilaterally symmetrical component contained in this potential (being smallest with almost unconsciously performed refixation saccades and smaller in trained than in naive subjects) appeared to be related mainly to the subjects' volitional effort. In addition, anticipatory and refixation saccades were preceded by an early, widespread contralateral negativity, which we relate to cortical activities that prepare, in general terms, action within or towards the hemifield containing the saccade goal. During the 60 msec before anticipatory saccades, a negativity occurred over the contralateral central lead, which may reflect neural activation in the frontal eye field (FEF) and premotor cortex. In contrast, regular saccades were preceded 30 msec before onset by a negativity over the contralateral parietal cortex, which probably reflects an activation of parietal visuo-motor neurons. No lateralization of the cortical potentials was observed before express saccades, which suggests that these saccades are generated in a reflex-like way mainly by subcortical mechanisms.


Cortical somatosensory magnetic responses in multiple sclerosis.

Somatosensory evoked magnetic fields (SEFs) to contralateral median and ulnar nerve stimulation were analyzed in 10 patients with multiple sclerosis and in 8 healthy controls. SEFs were recorded with a 24-channel SQUID gradiometer over both hemispheres. Seven patients showed abnormally large-amplitude SEF deflections at 60-80 msec; 5 of them had multiple lesions around lateral ventricles in magnetic resonance imaging. In 2 patients with plaques at the level of 3rd and 4th ventricles and medulla, the 30 msec responses were enlarged. The equivalent sources of 20 msec and 30-80 msec responses were in the primary hand sensorimotor cortex both in patients and in control subjects. The results suggest that early and middle-latency SEFs reflect parallel processing of somatosensory input. Recording of middle-latency evoked responses, electric or magnetic, may give additional information about the somatosensory function in multiple sclerosis.


Chemosensory event-related potentials in the investigation of interactions between the olfactory and the somatosensory (trigeminal) systems.

The aim of the study was to investigate the interaction of the olfactory and somatosensory systems in the perception of chemical stimuli. Stimuli were chosen so as to selectively activate the olfactory (hydrogen sulphide, H2S) and trigeminal (carbon dioxide, CO2) nerves. In addition, carvone was included as a stimulus with mixed properties. Thirty healthy volunteers participated in the experiments. Subjects rated the intensity of each of the stimulants when presented alone and as a component of binary mixtures. Chemosensory event-related potentials (CSERPs) were obtained from 5 recording positions. Analysis of the intensity ratings indicated that there was no difference between the 3 stimulants when used as single components. In binary mixtures intensity estimates of H2S were suppressed by CO2 and carvone. In addition, while estimates of CO2 were suppressed by carvone estimates of the latter were enhanced in the same mixture. CSERP data confirmed earlier findings with regard to the topographic distribution of amplitudes, i.e., if the olfactory system had been activated largest amplitudes were observed at position Pz, whereas activation of the trigeminal nerve produced largest amplitudes at Cz. Moreover, the suppression of CO2 estimates by carvone was reflected in a corresponding suppression of the CSERP amplitudes. In addition, when CO2 was mixed with H2S or carvone there was a decrease in the CSERP latency indicating interactions of both sensory systems in the time domain.


Evidence for terminal decline in the event-related potential of the brain.

Precipitous decline (terminal decline) characterizes changes in the elderly just prior to death. The neurological and temporal parameters of terminal decline are not defined. The present study compared sensitive changes in the brain event-related potential (ERP) in 7 subjects who died within 1 year of evaluation with matched controls. Cross-correlation of annual ERPs revealed disorganization within and between subjects in response to memory dependent conditions but not in response to a novel stimulus (oddball). Motor reaction time to targets was not sensitive to decline. The changes associated with impending death were restricted to decline in reliability of the ERP within 1 year of death.


Differential expression of basal and hydrocarbon-induced cytochrome P-450 monooxygenase and quinone reductase activities in subpopulations of murine epidermal cells differing in their stages of differentiation.

The activities of NAD(P)H-dependent quinone reductase (QR) and the cytochrome P-450 monooxygenases 7-ethoxycoumarin O-deethylase (7-ECD) and 7-ethoxyresorufin O-deethylase (7-ERD) were measured in four subpopulations of murine epidermal keratinocytes (MKs) that differed in their stages of differentiation. Noninduced per cell 7-ECD and 7-ERD activities were the lowest in basal cell MKs and progressively increased as the MKs underwent differentiation. In contrast, noninduced per cell QR activities in the three less differentiated MK subpopulations were very similar to one another and greater than the activities measured in the most differentiated subpopulation. Treatment of dorsal skin with 100 nmol of dibenz[a,c]anthracene (DB[a,c]A) increased CYPIA1 mRNA abundance and elevated 7-ERD activities to similar per cell levels in all MK subpopulations. This was achieved by differential inductions (200- to greater than or equal to 1850-fold) of 7-ERD in the different subpopulations. In contrast, QR induction by DB[a,c]A was similar (less than 3-fold) in all MK subpopulations. Consequently, the expressions of noninduced QR and 7-ERD activities in skin are regulated as a function of MK differentiation. However, the distributions of the noninduced activities of these two enzymes in MK subpopulations are the exact opposite. Furthermore, the relative inducibility of 7-ERD, but not QR, in skin is also regulated as a function of epidermal differentiation.


A 10 Hz "alpha-like" rhythm in the visual cortex of the waking cat.

Rhythms at about 10 Hz were recorded from the primary visual cortex of the cat (anterior part of area 18), with characteristics close to those of the alpha rhythm in man: frequency band (7-14 Hz), localization and reactivity to visual stimulation. Coherence analysis of this activity with the "mu" rhythms on the somatosensory cortex showed that although both types develop in the same overall behavioural situations (quiet waking and/or expectancy of an event to occur), they are independent.


Single photon emission computed tomography study of subclinical rhythmic electrographic discharge in adults.

Subclinical rhythmic electrographic discharge in adults (SREDA) is considered a benign EEG pattern of uncertain significance, although it may closely resemble an EEG seizure pattern. We investigated a 57-year-old man with a very lateralized epileptiform activity localized to the occipito-temporal areas of the right hemisphere. Neuropsychological tests, clonazepam injection and 99m Tc-HMPAO-SPECT were performed during the SREDA and compared to the interparoxysmal data, providing further evidence that SREDA cannot be considered as an epileptic pattern and that, in some instances, it may be related to chronic hypoxic/ischemic mechanisms.


Isotope archaeology: reading the past in metals, minerals, and bone.

The latest edition of the Oxford Dictionary (1989) defines archaeology as '... the scientific study of the remains and monuments of the prehistoric period'. It is not surprising, therefore, that modern archaeology draws as much as possible on scientific methods of investigation developed in other fields. In the last ten years the powerful method of quantitative isotope analysis has brought a new dimension to the examination of archaeological finds.


Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain.

We studied the inhibitory effect of non-phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143-1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine-phosphorylated peptides were synthesized using the N-(9-fluorenylmethoxycarbonyl)-O-dibenzylphosphono-L- tyrosine. The triphosphorylated peptide (1151-P3) and the non-phosphorylated peptide (1151-NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose-dependent and additive manner. When the receptor was pre-phosphorylated for 1 min with [gamma-32P]ATP, 1151-P3 decreased autophosphorylation to 60% of maximum, whereas 1151-NP had no further effect. In both non-activated and preactivated receptors, 1151-P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose-dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151-P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151-NP and 1151-P3 competition for the phosphorylation of a resin-immobilized 1151 peptide. While 1151-NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151-P3 caused a four-fold increase in the phosphorylation of 1151-NP--resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314-1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non-phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151-NP, but not of 1151-P3, thus suggesting a conformational change induced by the non-phosphorylated peptide. In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151-peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151-NP--resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.


Glutamatergic regulation of histamine release from rat hypothalamus.

A microdialysis method was used to study the effects of glutamate on the in vivo release of histamine from the anterior hypothalamic area of rats anesthetized with urethane. Infusion of 1 mM glutamate through a microdialysis probe increased histamine release to about 150% of the basal release. Infusion of N-methyl-D-aspartate (NMDA, 0.1 mM) caused a similar increase. Glutamate-evoked histamine release was completely blocked by D-(-)-2-amino-5-phosphonopentanoic acid (AP5, 0.1 mM), a specific antagonist of NMDA receptors. AP5 alone also reduced histamine release to about 60% of the basal level. Infusion of tetrodotoxin (100 nM) reduced histamine release to about 30% of the basal release, but had no effect on glutamate-evoked release. These results clearly indicate that glutamate enhances histamine release through NMDA receptors located on histaminergic nerve terminals, and suggest that there is a tonic glutamatergic regulation of this release.


Effect of resiniferatoxin on the isolated rabbit iris sphincter muscle: comparison with capsaicin and bradykinin.

The effect of resiniferatoxin on the isolated iris sphincter muscle of the rabbit was compared with the effects of capsaicin and bradykinin. The three compounds all contracted the sphincter muscle in a concentration-dependent manner. The contractions were inhibited by spantide II, a tachykinin antagonist. The concentration-response curve of resiniferatoxin was biphasic, yielding two EC50 values, one about 10,000 times lower than the other, the latter value being similar to that for capsaicin. The responses to resiniferatoxin, capsaicin or bradykinin were progressively reduced upon repeated application. The contraction evoked by either of the three compounds was not affected by thiorphan (an enkephalinase inhibitor) or captopril (an angiotensin-converting enzyme inhibitor). Pretreatment with capsaicin concentration dependently reduced the contractile response to a subsequent application of resiniferatoxin and vice versa. Bradykinin pretreatment reduced the resiniferatoxin response by about 50%; resiniferatoxin pretreatment completely abolished the response to bradykinin. Also, the contractile response to electrical stimulation was reduced concentration dependently by resiniferatoxin, capsaicin and bradykinin pretreatment. The response to electrical stimulation could be completely abolished by pretreatment with large doses of resiniferatoxin or capsaicin; pretreatment with large doses of bradykinin reduced the response by about 50%. Pretreatment with high concentrations of resiniferatoxin, or capsaicin, but not bradykinin, reduced the contractile response to the NK1 receptor agonist, [Sar9, Met(O2)11]SP (10(-8) M). The results suggest that the three compounds produce tachyphylaxis, mainly through partial (bradykinin) or complete (capsaicin, resiniferatoxin) exhaustion of the neuronal pool of releasable tachykinins although desensitization of tachykinin receptors may also contribute.


Interleukin-6 (IL-6) gene expression and secretion by cytokine-stimulated human retinal pigment epithelial cells.

Retinal and choroidal inflammatory lesions are important causes of visual loss, but the mechanisms regulating intraocular inflammation remain poorly understood. By virtue of its position at the blood-retina barrier, the retinal pigment epithelium (RPE) cells may be critical to the initiation and propagation of ocular inflammation. Previously we showed that cytokine-stimulated RPE cells produce interleukin-8, a well-defined chemotactic factor for neutrophils and lymphocytes. In this study, we found that human RPE cells stimulated by human recombinant interleukin-1-beta (rIL-1 beta) or tumor necrosis factor-alpha (rTNF-alpha) produce interleukin-6 (IL-6). Using a plasmacytoma proliferation assay, significant levels of IL-6 were found in media of RPE cells stimulated with either rIL-1 beta or rTNF-alpha for 4 hr. Progressive accumulation of IL-6 in media overlying stimulated RPE cells occurred over the subsequent 20 hr. IL-1 beta was a significantly more potent stimulator of RPE IL-6 production than TNF-alpha, RPE IL-6 production in response to each of these cytokines was also dose-dependent over a range of 20 pg to 20 ng ml-1. Specific anti IL-6 antibody, but not control immunoglobulin, neutralized RPE-derived IL-6 activity in the plasmacytoma proliferation assays. RPE IL-6 mRNA levels were detectable 1 hr after cytokine stimulation, plateaued within 8 hr in 24-hr assays, and demonstrated dose-dependent kinetics in 6 hr assays. Lipopolysaccharide failed to induce RPE IL-6 mRNA expression or RPE IL-6 production.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypertonic stress induces alpha B-crystallin expression.

Alpha B-crystallin, a major lens protein, was induced in primary cultures of dog lens epithelial cells and glomerular endothelial cells when they were grown under conditions of hypertonic stress. With Western blot analysis using a specific alpha B-crystallin antibody, we observed a significant increase in the concentration of alpha B-crystallin protein in cells grown for 4-6 days in media supplemented with 150 mM NaCl or 250 mM cellobiose. These supplements increased the osmolarity of the medium from 300 to 550-600 mosmol kg-1. Alpha B-crystallin mRNA was also increased reaching a maximum four-fold increase in lens and 16-fold increase in kidney cells within 1-2 days. These studies demonstrate a type of regulation of alpha B-crystallin expression in cells from lenticular and non-lenticular tissues.


An in situ perfused guinea-pig eye model for blood-ocular transport studies: application to amino acids.

A technique for in situ vascular eye perfusion (VEP) in the guinea-pig has been developed for measurements of the blood-ocular transport kinetics of substrates under controlled conditions of arterial inflow. The blood-aqueous and blood-vitreous barriers remained intact to the vascular space marker [3H]dextran (MW 70 kDa) with the perfusion pressure maintained between 80 and 100 mmHg. Several 3H-, 14C- or 35S-labeled amino acids, and 3H- or 14C-labeled sucrose (extracellular space marker) were used to validate the VEP model for transport kinetic studies. Multiple time-point graphic analysis was used to estimate the compartmental unidirectional blood-ocular transport constants, K(in), within the 20 min period of the VEP experiment. Blood-to-aqueous humor K(in) values for [3H]serine, [14C]N-methyl-alpha-aminoisobutyric acid (MeAIB) and [3H] or ]14C]sucrose were 3.57 +/- 0.38, 1.21 +/- 0.13 and 1.13 +/- 0.17 microliters min-1 g-1, respectively. The respective blood-to-lens K(in) values for labeled serine, MeAIB and sucrose were 1.71 +/- 0.19, 0.09 +/- 0.03 and 0.03 +/- 0.002 microliters min-1 g-1. The uptake of newly secreted amino acids from the aqueous humor in the lens followed the order: [35S]methionine greater than [3H]serine greater than or equal to [35S]cysteine greater than or equal to [3H]alanine greater than [14C]cycloleucine greater than or equal to [14C]MeAIB greater than or equal to [3H] or [14C]sucrose. The data indicate lack of a rapid blood-to-lens uptake of two model amino acids defining the A and L amino acid carriers.(ABSTRACT TRUNCATED AT 250 WORDS)


Photoreceptor-specific mRNAs in mice carrying different allelic combinations at the rd and rds loci.

Several retinal mRNAs encoding photoreceptor-specific proteins have been examined in congenic lines of mice carrying different allelic combinations at the rd and rds loci, to determine how mRNA expression is affected by the presence of both the rd and rds genes together or by the presence of one or two rd and rds alleles in the visual cells. Slot blots with retinal RNA from 9 to 30 days old C3H +/+, +/+; rd/+, +/+; rd/rd, +/+; +/+, +/rds; +/+, rds/rds; and the rd/rd, rds/rds double homozygous mutant mice were hybridized successively to several [32P]cDNA probes encoding proteins involved in phototransduction. The increases and decreases in the levels of mRNA coding for opsin, the alpha-subunit of transducin, 48 kDa protein and the beta-subunit of cGMP-phosphodiesterase in the heterozygous and homozygous rds and rd mouse retinas reflected the growth and degeneration of the photoreceptor cells. The heterozygous rd mouse (rd/+, +/+) retina expressed cGMP-phosphodiesterase beta mRNA levels halfway between those in the control (+/+, +/+) and homozygous (rd/rd, +/+) mice, indicating a possible dosage effect. The double homozygous mutant (rd/rd, rds/rds) showed intermediate levels between those observed in the two homozygotes for all mRNAs studied, suggesting a possible interaction between the rd and rds genes.


Cl- transport in frog retinal pigment epithelium.

Cl- transport across the retinal membrane of the frog retinal pigment epithelium was studied by means of double-barrelled Cl- selective microelectrodes. Three types of experiments were performed. In the first group of experiments, the ionic dependence of Cl- influx across the retinal membrane was studied. The intracellular Cl- activity was first decreased by perfusing the retinal side of the epithelium with low Cl- solutions (3.6 mM Cl-); then the perfusate was changed to high Cl- solutions (90.1 mM), and the resulting Cl- influx was studied. In these experiments, the combined presence of extracellular Na+ and K+ was a necessary condition for Cl- influx across the retinal membrane. This supports the hypothesis of Na+,K+,Cl- co-transport across this membrane. In a second group of experiments, the effect of furosemide was studied. Furosemide (100 microM) inhibited Cl- influx when the retinal extracellular Cl- concentration was increased from 3.6 to 90.1 mM. When administered to cells in steady state, furosemide in concentrations between 5 and 1000 microM decreased the intracellular Cl- activity. Michaelis-Menten analysis yielded a Ki for furosemide of 7 +/- 2 microM. The effect of furosemide on the intracellular Cl- activity required the combined presence of extracellular Na+ and K+. When the retinal extracellular K+ concentration was increased to between 0 and 10 mM, the furosemide-sensitive Cl- influx across the retinal membrane increased. Michaelis-Menten analysis yielded a half maximal stimulation at an extracellular K+ concentration of 0.5 mM. Stimulation of the epithelium with 1 mM cAMP and 0.5 mM IBMX reduced the effect of furosemide on the intracellular Cl- activity by 26%. In a third group of experiments, the effect of transepithelial currents on the intracellular Cl- activity was investigated. Currents that depolarized the choroidal membrane potential increased the intracellular Cl- activity; currents that hyperpolarized this membrane potential decreased the intracellular Cl- activity. These findings are compatible with conductive Cl- transport across the choroidal membrane. The apparent Cl- conductance of this membrane was estimated to be 0.59 mS cm-2. This represents 27% of the total conductance in the choroidal membrane. Administration of 1 mM cAMP and 0.5 mM IBMX caused a 21% increase in the apparent Cl- conductance of the choroidal membrane.


The influence of age and chronic restricted feeding on protein synthesis in the small intestine of the rat.

Rates of protein synthesis (measured in vivo) and growth of the small intestine were studied as a function of age in ad libitum fed (control) and chronic dietary-restricted rats. At weaning, the fractional rates of synthesis in the mucosal and muscularis externa and serosal layers of the small intestine of control animals were similarly high (90-100% per day). Although these rates subsequently declined with age in the muscularis externa and serosa, they remained constant in the mucosa. Restricted feeding (50% reduced intake), when imposed from weaning onwards, significantly extends the maximum life span of rodents. However, the change in nutritional status slows the accumulation of protein, RNA, and DNA in both layers of the small intestine. Although underfeeding did not prevent the age-related fall in muscularis externa and serosal protein synthesis, significantly higher rates (both fractional and per ribosome) were found when compared age for age with controls. Mucosal fractional synthetic rates were similarly increased by the reduced food intake. These changes in protein turnover in the small intestine are consistent with the higher rates of whole body turnover previously observed in chronically underfed rats.


Anatomical and functional compartmentalization of the subparafascicular thalamic nucleus in the rat.

The localization and the transmitter phenotype of subparafascicular thalamic nucleus (Spf) neurons projecting to the inferior colliculus (IC) and to the spinal cord (Sp) were studied by using a retrograde fluorescent double labeling technique, and a combined technique of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD). The cell population of Spf-IC neurons was totally differentiated from that of Spf-Sp neurons which have been reported to be dopaminergic. The former were densely distributed, small to medium sized cells and localized in the central portion of the Spf, while the latter were sparsely distributed, large cells and localized in the marginal portion of the Spf. Spf-IC neurons were completely devoid of TH immunoreactivity and, instead, approximately half of them showed GAD immunoreactivity. From these findings, it is concluded that the Spf is distinctly compartmentalized by the presence at least two separate neuronal subpopulations, which are distinguishable in terms of their cell size, distribution patterns, transmitter phenotypes and trajectories.


Perturbation of cellular energy state in complete ischemia: relationship to dissipative ion fluxes.

Loss of cellular ion homeostasis during anoxia, with rapid downhill fluxes of K+, Ca2+, Na+ and Cl-, is preceded by a slow rise in extracellular K+ concentration (Ke+), probably reflecting early activation of a K+ conductance. It has been proposed that this conductance is activated by either a rise in intracellular calcium concentration (Cai2+), or by a fall in ATP concentration. In a previous study from this laboratory (Folbergrová et al. 1990) we explored whether the early activation of a K+ conductance could be triggered by a rise in Cai2+. To that end, labile metabolites and phosphorylase a, a calcium sensitive enzyme, were measured after 15, 30, 60 and 120 s of complete ischemia ("anoxia"). In the present study, we investigated whether brief anoxia is accompanied by changes in ATP/ADP ratio, or in the phosphate potential, which could cause activation of a K+ conductance. To provide information on this issue, we added a group with 45 s of anoxia to the previously reported groups, and derived changes in intracellular pH (pHi). This allowed calculations of the free concentrations of ADP (ADPf) and AMP (AMPf) from the creatine kinase and adenylate kinase equilibria, and hence the derivation of ATP/ADPf ratios. In performing these calculations we initially assumed that the free intracellular Mg2+ concentration remained unchanged at 1 mM. However we also explored how a change in Mgi2+ of the type described by Brooks and Bachelard (1989) influenced the calculation. The results showed that ADPf must have risen to 150-200% of control within 15 s, and to 330-350% of control within 45 s of anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)


Operative bypass for incurable cancer of the head of the pancreas.

A series of 83 patients with incurable cancer of the pancreatic head were analysed. Obstructive jaundice required surgical intervention in all of them. Different biliary decompression operations, either single or with concomitant prophylactic gastro-enterostomy were constructed in different ways. From our study we conclude: a single bypass, using the gallbladder and anastomosed with the jejunum is preferred in those cases where life expectation is estimated to be less than 3 months. Delayed gastric emptying after gastro-enterostomy is a frequent complication. Double bypasses are recommended only in those cases where duration of life is estimated at more than 3 months.


The effect of estrogen level on glucose-induced changes in serum insulin-like growth factor binding protein-1 concentration.

To investigate the regulation of insulin-like growth factor binding protein-1 (IGFBP-1) concentration during ovarian stimulation. A prospective study of patients undergoing in vitro fertilization treatment. Infertility unit at the University Central Hospital of Oulu, a tertiary referral center. Sixteen healthy, regularly menstruating lean tubal infertility patients. Oral glucose tolerance test was performed first in a hypoestrogenic state after suppression by long-term gonadotropin-releasing hormone (GnRH) agonist and, second, in a hyperestrogenic state after stimulation by human menopausal gonadotropins. Serum concentrations of IGFBP-1, insulin-like growth factor I (IGF-I), insulin and sex hormone-binding globulin were measured before and 2 hours after glucose administration. Before and after glucose administration, the serum IGFBP-1 concentrations were significantly higher in the hyperestrogenic state (estradiol [E2] level 3.5 +/- 0.57 nmol/L) after ovarian stimulation than in the GnRH-analogue-induced hypoestrogenic state before the gonadotropin treatment (E2 level 0.10 +/- 0.02 nmol/L). On both occasions glucose-induced hyperinsulinemia caused a significant decrease in the circulating IGFBP-1 levels, whereas the IGF-I levels remained unchanged. There was a significant correlation between E2 and the insulin-suppressed IGFBP-1 level. The sum of follicular diameters correlated positively with the serum IGFBP-1 concentration. Gonadotropin-induced hyperestrogenism is related to elevated serum IGFBP-1 levels, either via estrogen-stimulated synthesis or via increased contribution from multiple follicles. Glucose-induced hyperinsulinemia suppresses serum IBFBP-1 concentration equally both in the hypoestrogenic and hyperestrogenic states. Because of similar IGF-I levels, it is likely that the biological activity of IGF-I is different before and after gonadotropin stimulations.


Intrauterine pregnancy in a patient with a sole remaining tube after local treatment of tubal pregnancy with hyperosmolar glucose.

A spontaneous IUP occurred 8 months after laparoscopic instillation of hyperosmolar glucose solution into a tubal pregnancy in a patient with a sole remaining tube. This is the first unequivocal proof of intact tubal function after treatment of a tubal pregnancy with local hyperosmolar glucose.


The effect of dextran on the pharmacokinetics of lignocaine during epidural anaesthesia.

The clinical significance of combining local anaesthetics with dextran is controversial. Although a number of investigators have found that dextran can prolong the action of local anaesthetics, others have not been able to demonstrate any significant change in the duration of anaesthesia. A study was carried out to investigate the effect of dextran on the pharmacokinetic behaviour of lignocaine during epidural anaesthesia in 20 adult male patients, who were randomly allocated to two treatment groups: group 1 (n = 10) was given lignocaine-dextran; group 2 (n = 10) was given lignocaine-saline. The results of the study demonstrated that the addition of dextran to epidural lignocaine significantly slowed systemic absorption of lignocaine as indicated by a smaller absorption rate constant, a reduced peak plasma concentration (Cmax), a delayed time to reach Cmax. and a smaller area under the concentration-time curve. The observed findings suggest that dextran reduces vascular uptake of lignocaine from epidural space and that it may prolong the duration of action. The significance of these findings on systemic toxicity, dosage and onset of action of lignocaine need to be investigated.


Nitric oxide as a secretory product of mammalian cells.

Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.


Bovine Escherichia coli of serotypes O55:H4 and O55:H21 which produce CNF2 express P fimbriae and Vir surface antigen, respectively.

We have characterized the toxic and adhesive properties of Escherichia coli strains producing the second type of cytotoxic necrotizing factor (CNF2) and belonging to the classic enteropathogenic serogroup O55. Bovine CNF2 strains of serotype O55:H4 express P fimbriae as do pyelonephritic Escherichia coli that cause infections in humans. In contrast, strains of serotype O55:H21 which produce CNF2 from bovine origin possess the Vir surface antigen. One human strain of serotype O55:H- was positive for production of CNF2, but was negative for the two adhesive factors and for mannose-resistant haemagglutination.


Synthesis of unusual thick pili by Escherichia coli of EPEC serogroup O119.

Unique very thick pili were found in varying numbers on cells of five out of 11 clinical isolates of enteropathogenic Escherichia coli belonging to the classic EPEC serogroup O119. They were approximately 12.5 nm in diameter, which is thicker than any other known E. coli pilus type. Analysis of the plasmid profiles of the O119 isolates showed that one strain was plasmid-free while the remainder contained numerous plasmids with a wide range of sizes. The thick pilus determinants were located on a 140-kb non-transferrable plasmid. They were not associated with adherence or a putative 90-kb enteroadherence factor plasmid.


Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing.

The primary structures of the 23S ribosomal RNA genes of Bacillus anthracis and an emetic strain of Bacillus cereus were determined by direct sequencing of enzymatically amplified chromosomal DNA. The 23S rRNA gene sequences of B. anthracis and B. cereus were found to be almost identical and showed only two differences (a single nucleotide change, and a single base insertion in B. cereus). The feasibility of using PCR-direct sequencing for the rapid sequence determination of large-subunit rRNA genes is demonstrated.


Apolipoprotein E polymorphism in a Danish population compared to findings in 45 other study populations around the world.

Apolipoprotein E (apoE) phenotypes were determined in a random sample of 466 Danish men born in 1948. The frequencies of the common alleles of the apoE gene were (with 95% confidence intervals) epsilon 2 = 0.085 (0.068-0.105), epsilon 3 = 0.741 (0.712-0.769), and epsilon 4 = 0.174 (0.150-0.200). These frequencies were compared to findings in 45 other study populations around the world (n greater than 100). The Danish population was found to cluster with populations from Iceland, Norway, Iceland, Scotland, the Netherlands, Germany, France (Paris), and Caucasian populations in Canada and the USA. The compiled data further show that dissimilarities in apoE allele frequencies among Caucasian populations are comparable to dissimilarities between some Caucasian and Asian populations. Notably, the frequency of epsilon 4 appears to be higher in northern regions of Europe (the Nordic countries, Scotland, Germany, and the Netherlands) than in southern regions (Switzerland, Tyrol, France [Nancy], Italy, and Spain).


Inhibition effects of KRDS, a peptide derived from lactotransferrin, on platelet function and arterial thrombus formation in dogs.

KRDS (Lys-Arg-Asp-Ser), a tetrapeptide from human lactotransferrin, was tested for its effects in vitro on dog platelet function and in vivo on femoral arterial thrombus formation in dogs. KRDS inhibited ADP (8 microM)-induced platelet aggregation (IC50: 350 microM) and arachidonic acid (2 mM)-induced thromboxane B2 generation (IC50: 175 microM). In addition, the thrombin (0.2 U/ml)-induced serotonin release was inhibited by KRDS (IC50: 525 microM) and the expression of alpha-granule membrane protein (GMP-140) was also inhibited (IC50: 350 microM). The results show that KRDS is an inhibitor for platelet aggregation and secretion to which the inhibition is more potent. Meanwhile, in the experiment of arterial thrombosis in dogs, KRDS (5 microM/kg) and 125I-SZ-51 (a monoclonal antibody against GMP-140) were injected before operation and immediately after the thrombus formation, respectively. In the KRDS group, the weight of removed thrombi was reduced to 50% of that in controls and the radioactivity per mg of labeled thrombi to 33.3% while in blood the radioactivity increased 2 times that in controls at the 4th hour after the injection of 125I-SZ-51. The radioactivity ratio between removed thrombi and blood was only 16% of that in controls. These results indicate that KRDS can inhibit thrombus formation in vivo and is a promising antithrombotic agent.


[Acute trichinosis. Epidemics of 166 cases in Delicias City, Chih. Diagnosis with tissue compression and staining].

The purposes of this report are: a) To show outstanding clinicopathological characteristics from the largest outbreak of trichinosis in México. b) To describe a modification to the traditional trichinoscopy through the use of compressed muscle stained with ordinary dyes an immediate observation; we have called the procedure tissular compression and dyeing technique (TCDT). The outbreak occurred in Delicias, Chihuahua, and affected 166 inhabitants who ate a not well cooked pork sausage. All patients showed malaise, 85.5 percent myalgias, and 72.7 percent facial edema; leucocytosis was seen in 75 percent of the patients, 60 percent with eosinophilia. Electromyography was performed in 21 patients in the acute phase of the illness and it was found abnormal in 85.7 percent. Only one patient died (0.6%). From 59 patients who were considered very ill, muscle biopsies were taken, and the conventional histopathological study showed larvae in 88.1 percent. TCDT demonstrated worms in 90.3 percent out of 47 biopsies; larvae were seen free and into muscle fibers with or without capsule cells. In conclusion TCDT is a simple, fast and easy reproducible procedure.


[Treatment of chronic hepatitis B. Part 1: Critical overview of current results of studies].

Critical appraisal of presently available results of studies on the treatment of chronic hepatitis B. Even the controlled studies on the treatment of chronic hepatitis B carried out in recent years, in particular involving the use of interferons, possibly in combination with glucocorticosteroids, real methodological shortcomings that, in part, are due to the characteristics of chronic hepatitis, such as variations in its evolution, inadequate information provided by individual evolution-related parameters and failure to take account of patient variables. Moreover, the spontaneous evolution of the disease is difficult to predict, and a definitive assessment of the results of treatment always requires longterm follow-up. Despite the shortcomings of the individual studies, treatment with interferon represents a great advance in the therapy of chronic hepatitis B. However, the high percentage of treatment failures makes the continued search for new therapeutic modalities necessary.


Evaluation of keratinolytic potential of some fungal isolates from gelatin factory campus.

During hair degradation, majority of organic sulphur was oxidized to inorganic sulphate and thiosulphate by four fungal isolates (Cylindrocarpon lichenicola, Graphium cuneiferum, Microsporum gypseum, and M. fulvum) from gelatin factory soil. Inorganic thiosulphate, an unusual metabolite, was regularly detected in the culture filtrates of all fungi, although in less amounts. Maximum quantity (44 micrograms/ml) was released by G. cuneiferum on 50th day of incubation. All four fungi showed significant extracellular keratinase activity on human hair. Sulphydryl compounds were present in low amounts throughout the experiment. Detection of inorganic sulphate and thiosulphate with significant release of total protein and keratinase and changes in alkalinity, established the role of sulphitolysis and peptidolysis during keratin biodegradation by fungi which ultimately results in complete keratin degradation.


Anti-implantation role for substance P and neurotensin in rat.

Substance P (SP) and neurotensin (NT), two structurally related peptides with contrasting biological actions, have been shown to have some role in peripheral reproductive processes. Intrauterine microinjection of SP or NT on day 4 or 5 of pregnancy in the rat significantly reduced the number of viable fetuses, weight and glycogen content of the uterus. The number of viable fetuses, uterine weight or glycogen content were not modified when SP/NT was microinjected on day 8, 9, 10 or on day 14, 15 and 16. The results indicate that the peptides possibly exert a direct local alteration in uterine vascular permeability causing failure in implantation.


Progression of human cutaneous melanoma is associated with loss of expression of c-kit proto-oncogene receptor.

Mutations at the white spotting (w) locus in mice have deleterious effects on germ cells, melanocytes and hematopoietic stem cells. The w locus encodes the c-kit tyrosine-kinase receptor whose ligand is the product of the SI locus. Using monoclonal antibodies (MAb(s)) to the extracellular domain, we have evaluated the expression of c-kit in normal and transformed melanocytes. This cell lineage synthesizes a receptor with a mw of 145 kDa. The gene product is expressed in epidermal melanocytes and in a fraction of nevocytic and blue nevi. In primary melanomas, loss of the receptor is observed in more invasive lesions. Only 30% of the metastatic lesions express detectable levels of the receptor. These findings demonstrate that the c-kit product is down-regulated in melanocytes following malignant transformation. The functional relevance of this modulation remains to be evaluated.


The effect of endocrine therapy with medroxyprogesterone acetate, 4-hydroxyandrostenedione or tamoxifen on plasma concentrations of insulin-like growth factor (IGF)-I, IGF-II and IGFBP-1 in women with advanced breast cancer.

Tamoxifen treatment of women with advanced breast cancer has previously been reported to reduce plasma insulin-like growth factor-type I (IGF-I) concentrations. In this study we have examined the effect of treatment with Tamoxifen, medroxyprogesterone acetate (MPA) or 4-hydroxyandrostenedione (4-OHA) on plasma IGF-I and IGF-II concentrations. As IGF-binding proteins (IGFBPs) can modulate the biological effects of IGF-I, plasma IGFBP-I levels were also measured. Treatment with Tamoxifen for 2 weeks resulted in a small, but significant, decrease in IGF-I levels, but increase in the plasma concentration of IGFBP-I. In contrast, treatment with MPA increased levels of IGF-I, but significantly reduced plasma IGFBP-I concentrations. Treatment with 4-OHA had no significant overall effect on plasma IGF-I or IGFBP-I levels, although changes were detected for some subjects. Plasma IGF-II concentrations were not altered by treatment with Tamoxifen, MPA or 4-OHA. It is concluded that although treatment with Tamoxifen or MPA produced significant changes in plasma IGF-I concentrations, any physiological effects of such changes are likely to be modulated by the corresponding alterations in plasma IGFBP-I concentrations.


Phenotype-specific "tissue" transglutaminase regulation in human neuroblastoma cells in response to retinoic acid: correlation with cell death by apoptosis.

Neuroblastomas in culture are characterized by the presence of 2 morphologically and biochemically distinct phenotypes (i.e., neural "N-type" and flat substrate-adherent "S-type") which undergo transdifferentiation. Human neuroblastoma SK-N-BE(2) cells differentiate toward a neural phenotype upon retinoic acid (RA) treatment. However, we recently showed that, during the RA treatment, a subset of SK-N-BE(2) cells undergo apoptosis; these cells specifically express a high "tissue" transglutaminase (tTG) level. This study was undertaken to investigate the cellular and molecular basis of the action of retinoic acid on apoptosis in human neuroblastoma cells. As a biochemical marker of the phenomenon we studied the tTG gene expression in the parental line SK-N-BE(2) and in 2 clones which stably express neuroblastic [BE(2)-M17] and substrate-adherent [BE(2)-C] features, respectively. Data showed a differential phenotype-specific regulation of tTG gene expression. In fact, RA treatment enhanced tTG expression and apoptotic index in the flat substrate-adherent variant, whereas, in cells expressing the neural phenotype, very low tTG expression and apoptosis were found. Northern-blotting analysis revealed that the substrate-adherent cells had a basal 3-fold higher level of tTG mRNA. An increase in tTG mRNA major transcript levels (3.7 kb) occurred within a few hours of exposure to RA in both the phenotypic variants. By contrast, tTG protein level was very low in the cell expressing the neuronal phenotype, even after prolonged exposure to RA. Immunohistochemical analysis indicated that tTG protein, in addition to mature apoptotic cells, was specifically localized in the flat substrate-adherent variant both in the wild-type and in the BE(2)-C clone. These findings suggest that the ability to undergo apoptosis in the neuroblastoma cells is associated with the expression of a non-neuronal neuroectodermal (substrate-adherent cells) immature phenotype.


Schedule-dependent effects of two consecutive, divided, low doses of actinomycin D on translocation of protein B23, inhibition of cell growth and RNA synthesis in HeLa cells.

The effects of 2 consecutive, divided, low doses of actinomycin-D (Act-D) on cellular localization of protein B23, inhibition of cell growth, RNA synthesis and colony formation were studied in HeLa cells. The second dose of Act-D was administered at various times after removal of the first dose. One short exposure of HeLa cells to Act-D had previously been shown to induce "reversible" translocation of protein B23, inhibition of cell growth, and RNA synthesis. Relocalization of protein B23 from the nucleoplasm to nucleoli as well as "reversible" inhibition of cell growth and RNA synthesis were still observed in cells that had been treated with a second dose of Act-D administered as early as 0-2 hr or as late as 30 hr after removal of the first dose of Act-D. In contrast, no relocalization of protein B23 from the nucleoplasm to nucleoli was observed in cells that had been treated with a second dose of Act-D administered 9 hr after removal of the first dose. A second exposure to Act-D, administered 9 hr after removal of the first dose, caused irreversible inhibition of cell growth and RNA synthesis; a significant inhibitory effect on colony formation was also observed. RNA synthesis in HeLa cells after 2 sequential exposures to Act-D was further analyzed by 1% agarose gel electrophoresis. There were higher-molecular-weight bands above 28S RNA, which may be the 45S and 32S RNA, observed in the controls and in the cells that had been exposed to Act-D treatment once or in the cells that underwent Act-D exposure twice, in which the second dose was administered as early as 0-2 hr or as late as 30 hr after removal of the first dose. These high-molecular-weight bands were not observed in the cells that underwent Act-D exposure twice, in which the second dose was administered 9 hr after removal of the first. These results indicated that cells at different stages of inhibition or that have recovered from the first exposure to Act-D respond differently to the second short Act-D exposure.


FK-506-binding proteins from streptomycetes producing immunosuppressive macrolactones of the FK-506 type.

FK-506-binding proteins (FKBPs), which in T cells are supposed to mediate the immunosuppressive effects of the compounds FK-506 and rapamycin, have been isolated from Streptomyces chrysomallus, S. hygroscopicus subsp. ascomyceticus, and S. hygroscopicus. The latter two strains are producers of ascomycin (the ethyl analog of FK-506) and rapamycin, respectively. Like the 12-kDa FKBP in eukaryotic organisms such as humans, bovines, and Saccharomyces cerevisiae, or the FKBPs from gram-positive streptomycetes are peptidyl-prolyl-cis-trans isomerases. Inhibition studies using FK-506, rapamycin, or ascomycin, revealed inhibition of the peptidyl-prolyl cis-trans isomerase activity of the proteins at the nanomolar level, which is in the same range as with eukaryotic FKBPs. The M(r)s of the various FKBPs were 13,500 to 15,000, and they had the same pI of approximately 4.5. The N-terminal sequences of the three FKBPs were nearly identical in the first 20 amino acids. The amino acid sequence deduced from the gene sequence of S. chrysomallus gave a polypeptide of 124 amino acids. The homologies to FKBPs from humans, S. cerevisiae, and Neurospora crassa were 38, 39, and 50% identity in relevant positions, respectively. Significant homology of 38% was also seen with the C-terminal halves of bacterial protein surface antigens like the Mip protein of Legionella pneumophila and the 27-kDa Mip-like protein of Chlamydia trachomatis. In addition, two more open reading frames in Pseudomonas aeruginosa and Neisseria meningitidis of unknown function show regions of homology to the S. chrysomallus FKBP. In contrast to fungi, streptomycetes are resistant to macrolactones. Ascomycin-producing S. hygroscopicus subsp. ascomyceticus excretes the compound almost quantitatively into medium, which indicates that the organism has an efficient self-protection mechanism against its own secondary metabolite.


Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength. Detergent extraction of carrot cells removed the receptor to which the bacteria bind. The extract was found to contain a vitronectin-like protein. These results suggest that A. tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.


A switch in cytokeratin expression and intermediate filament organization associated with epithelial stratification.

Low density gingival epithelial cells were cultured on the side of glass slides facing rat's tail collagen lattices. Under these conditions and in the presence of physiological level of calcium, colony formation was enhanced and stratification was slowed down. The strong attachment of the cells to glass slides permitted immunocytochemical examination of cytokeratin (CK) expression and their organization within individual cells during the different stages of epithelial maturation. The present results showed that during the stage of cell migration and colony formation, the cells express the same set of cytokeratins (basal cell marker 14, simple epithelial markers 8, 18 and 19, and marker of hyperproliferation 16) which forms a well-defined network of organized filaments. At the stratification stage, the filament network became dense by the additional expression of the markers of differentiation in non-keratinized stratified epithelia (CK 4 and 13). These appeared once individual cells started to overlap the basal cells, a period during which the cell-temporarily changed morphology. Whilst the suprabasal cells exhibited dense filament network labelled for CK 4 and 13, the density of labelled filaments for CK 14, 8 and 18 was much lower, indicating that these cells contained newly-formed filaments lacking the basal and simple epithelial keratins. The simple epithelial cytokeratins became weakly labelled in older cultures. The uncoupling of paired expression of cytokeratins 4 and 13 was observed in non-colony forming aged cells. This provides an example of altered program of cytokeratin expression during epithelial maturation.


Insulin-like growth factor binding protein (IGFBP-3), an inhibitor of serum growth factors other than IGF-I and -II.

Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-I as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-IGF-I IgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3.


Granulocyte-macrophage colony-stimulating factor and steel factor induce phosphorylation of both unique and overlapping signal transduction intermediates in a human factor-dependent hematopoietic cell line.

Steel factor (SF), the ligand for the proto-oncogene c-kit, acts synergistically with GM-CSF or IL-3 to support the growth of normal human hematopoietic progenitor cells. We examined the effects of SF on GM-CSF or IL-3 induced proliferation of a human factor-dependent cell line, MO7. SF supported MO7 cell proliferation as well as IL-3 or GM-CSF alone, and its addition dramatically enhanced (three- to sixfold) maximal GM-CSF or IL-3 stimulated proliferation. SF did not increase the number or affinity of cell surface GM-CSF receptors. We examined several early events of signal transduction in an effort to elucidate the biochemical mechanisms of synergy of these factors. Since each of these three cytokines is believed to function in part through activation of a tyrosine kinase, we examined their effects on cellular phosphotyrosine containing proteins. Each cytokine induced rapid, transient, and concentration dependent tyrosine phosphorylation of a number of substrates. For GM-CSF and IL-3, these phosphoproteins were indistinguishable (150, 125, 106, 93, 80, 79, 73, 44, 42, and 36 kDa), while SF induced major or minor tyrosine phosphorylation of 205, 140-150, 116, 106, 94, 90, 80, 79, 73, 44, 42, 39, 36, 32 kDa phosphoproteins. Two other signal transduction intermediates known to be phosphorylated and activated by GM-CSF and IL-3, the 70-75 kDa Raf-1 kinase, and p42 mitogen-activated protein kinase-2 (MAPK), were also phosphorylated by SF. Combinations of GM-CSF or IL-3 with SF did not further increase the phosphorylation of Raf-1 or p42 MAPK when compared to any of the factors alone. In contrast SF, but not GM-CSF or IL-3, induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). These results indicate that SF and GM-CSF/IL-3 have partially overlapping effects on early signal transducing events, as well as striking differences, such as tyrosine phosphorylation of PLC-gamma. This cell line should provide a useful model system to investigate the complicated process of hematopoietic growth factor synergy.


Ovulation induction increases serum levels of insulin-like growth factor binding protein 1.

The effect of ovulation induction on serum insulin-like growth factor binding protein 1 (IGFBP-1) level in relation to sex hormone binding globulin (SHBG) levels was evaluated. Serum samples were collected 8 to 12 days after ovulation from 26 women undergoing ovulation induction with clomiphene citrate (CC), and from 58 women treated with CC in combination with human menopausal gonadotropin (hMG) and human chorionic gonadotropin (hCG). In addition, serum samples were obtained from 63 spontaneously ovulating women and from 12 women during an anovulatory cycle. Luteal phase serum IGFBP-1 levels were 4.22 +/- 2.95 micrograms/L (P less than .05) in the CC group and 7.31 +/- 6.13 micrograms/L (P less than .001) in the CC/hMG/hCG group as compared to unstimulated ovulatory cycles (2.64 +/- 2.52 micrograms/L). No significant difference in IGFBP-1 levels was seen between spontaneously ovulatory and anovulatory cycles. The serum IGFBP-1 levels correlated positively to SHBG levels (r = .52, P less than .001). The data show that ovulation induction increases serum IGFBP-1 levels in parallel to SHBG levels, indicating that ovarian stimulation, which results in increased steroid hormone production, also induces changes in other factors known to modulate steroid hormone actions.


Receptor phenotype underlies differential response of hepatocytes and nonparenchymal cells to heparin-binding fibroblast growth factor type 1 (aFGF) and type 2 (bFGF).

Heparin-binding fibroblast growth factors (HBGF) have been implicated in the regeneration of both parenchymal and nonparenchymal cells of the liver. The response to and phenotype of hepatocyte receptors for HBGF-1 (acidic fibroblast growth factor) and HBGF-2 (basic fibroblast growth factor) were compared to keratinocytes, fibroblasts, and endothelial cells. HBGF-1 stimulated DNA synthesis in hepatocytes, keratinocytes, fibroblasts, and endothelial cells whereas activity of HBGF-2 was limited to fibroblasts and endothelial cells. HBGF-2 antagonized the mitogenic activity of HBGF-1 for hepatocytes and keratinocytes. Hepatocytes and keratinocytes exhibited both high- and low-affinity, nonmatrix receptor sites for HBGF-1, but only low-affinity sites for HBGF-2. The mesenchymal cells displayed only high-affinity sites for both HBGF-1 and HBGF-2. Northern blot and immunochemical analysis revealed that the expression of HBGF receptor genes bek and flg are partitioned between normal hepatocytes and nonparenchymal cells, respectively. Expression of epithelial cell-specific, mesenchymal cell-derived HBGF-7 (keratinocyte growth factor) mRNA in regenerating liver tissue was undetectable relative to HBGF-1. The results support a multifunctional role of HBGF-1 acting through different receptor phenotypes in hepatocyte and nonparenchymal cells during liver regeneration.


Neutrophil adhesion to TNF alpha-activated endothelial cells potentiates leukotriene B4 production.

Since adhesion of neutrophils (PMN) to endothelial cells may influence PMN activation responses, we examined whether adhesion of PMN to TNF alpha-activated human umbilical vein endothelial cells (HUVEC) stimulates leukotriene B4 (LTB4) production. Endothelial adhesivity towards PMN increased after HUVEC pretreatment with TNF alpha for 4 h. LTB4 production increased markedly in response to stimulation with arachidonic acid (20 microM) when PMN were added to the hyperadhesive HUVEC. In contrast, stimulation of PMN in suspension did not potentiate LTB4 production. LTB4 production persisted when PMN were applied to TNF alpha-pretreated HUVEC fixed with 1% paraformaldehyde excluding the possibility that metabolic activity of endothelium participates in this response. PMN adhesion to plastic and gelatin also enhanced LTB4 indicating that adhesion was a critical event in inducing LTB4 production. We used monoclonal antibodies (mAb) to adhesion molecules on endothelial cells (i.e., endothelial leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1)) or on PMN (CD18) to assess the role of PMN adhesion to the activated endothelium on LTB4 potentiation. Both anti-ELAM-1 mAb and anti-ICAM-1 mAb inhibited PMN adhesion (by 55 and 41%, respectively) as well as LTB4 production (by 65 and 50%, respectively). Anti-CD18 mAb also reduced the adhesion (65%) and the LTB4 production (66%). Furthermore, combination of anti-ELAM-1 mAb (H18/7) and anti-ICAM-1 mAb (RR1/1) or of anti-ELAM-1 mAb (H18/7) and anti-CD18 mAb (IB4) had an additive effect in inhibiting both PMN adhesion as well as LTB4 production. PMN adherence to immobilized recombinant soluble rELAM-1 or rICAM-1 also increased LTB4 production, which was prevented with relevant mAbs. However, neither rELAM-1 nor rICAM-1 stimulated LTB4 production of PMN in suspension. We conclude that PMN adhesion to TNF alpha-stimulated endothelial cells enhances LTB4 production by PMN, a response activated by binding of PMN to expressed endothelial cell surface adhesion molecules.


Current status of HIV therapy: I. Antiretroviral agents.

The clinician's armamentarium is no longer limited to zidovudine as the only antiretroviral agent that enhances both quality and length of life. ddI has shown clinical benefit in patients previously treated with zidovudine. Recently, the combination of zidovudine and ddC has been approved on the basis of surrogate marker activity; its clinical role awaits results of ongoing trials.


Optimization of the separation of the Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides.

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 x 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10-100 mg.


Interleukin-7 is a growth factor for Sézary lymphoma cells.

Sézary syndrome is a cutaneous T cell lymphoma characterized by infiltration of the skin by CD4+ cells. These cells generally respond poorly to mitogens and T cell activators. We have studied the action of IL1 to IL4, IL6, and IL7 on the proliferation of Sézary cells from 12 patients. With the exception of IL2 and IL7, the cytokines studied had no proliferative effect on these cells. Whereas IL2 had only a low proliferative capacity (two- to threefold increase) on peripheral blood mononuclear cells, recombinant IL7 constantly induced a very significant (3-40-fold increase) proliferative response, and was used successfully to generate cell lines in three out of eight cases. Growth of Sézary cell lines was shown to be strictly dependent on IL7, and after 2-5 wk of culture presented a switch to a homogeneous phenotype CD3+4+8-7- (except for one line that remained CD7+), with a typical morphology of Sézary cells. Their tumoral origin was demonstrated by the expression of the same T cell receptor-beta gene rearrangement as the patients' T cells. Importantly, cultured normal epidermal keratinocyte supernatants could support the growth of our Sézary lines. Furthermore, the proliferative activity contained in these supernatants was completely blocked by a monoclonal anti-IL7 antibody. These results suggest that IL7 may, therefore, represent an important cytokine in the physiopathology of cutaneous T cell lymphoma.


Inhibition of the complement membrane attack complex by the galactose-specific adhesion of Entamoeba histolytica.

The human complement system is an important early host defense against infection. Entamoeba histolytica activates the complement system but is resistant to killing by complement C5b-9 complexes deposited on the membrane surface. Our aim was to identify components of the amebic plasma membrane that mediate resistance to human complement C5b-9 by screening for neutralizing monoclonal antibodies. A monoclonal antibody was identified that abrogated amebic resistance to C5b-9, and the mAb was shown to recognize the parasite's galactose-specific adhesin. The purified adhesin bound to C8 and C9 and conferred C5b-9 resistance to sensitive ameba upon reconstitution; these activities of the adhesin were inhibited by the antiadhesin mAb. The E. histolytica adhesin shared sequence similarities and antigenic cross-reactivity with CD59, a membrane inhibitor of C5b-9 in human blood cells, suggesting both molecular mimicry and shared complement-inhibitory functions.


Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells.

Accumulation of monocyte-derived foam cells in focal areas of the arterial intima is one of the key events in early atherogenesis. We have examined the effect of lysophosphatidylcholine (lyso-PC; lysolecithin), a major phospholipid component of atherogenic lipoproteins, on the expression of adhesion molecules for monocytes, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), in cultured human and rabbit arterial endothelial cells. Cultured rabbit aortic endothelial cells treated with lyso-PC showed increased mRNA and cell surface expression of VCAM-1 and ICAM-1, which was associated with increased adhesion of monocytes and monocyte-like cells (THP-1, U937). In cultured human iliac artery endothelial cells, lyso-PC similarly induced both VCAM-1 and ICAM-1, whereas in umbilical vein endothelial cells only ICAM-1 was up-regulated. In all endothelial cells examined, the effect of lyso-PC on E-selectin (endothelial-leukocyte adhesion molecule-1) expression was negligible, thus differentiating this stimulus from other endothelial activators, such as interleukin 1, tumor necrosis factor, or lipopolysaccharide. We conclude that lyso-PC can selectively induce VCAM-1 and ICAM-1 in arterial endothelial cells and that this action, in addition to its monocyte chemoattractant activity, may play an important role in monocyte recruitment into atherosclerotic lesions.


Recognition by recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on human thyroid peroxidase.

To characterize the nature of thyroid peroxidase (TPO) autoantibodies present in the sera of patients with autoimmune thyroid disease, we cloned three IgG1/kappa Fab fragments which bind 125I-TPO. This was accomplished by the molecular cloning and expression in bacteria of IgG gene fragments from B cells infiltrating the thyroid of a patient with Graves' disease. The three Fab fragments (SP2, SP4, and SP5) are coded for by a common heavy chain (VH1, D, JH3) and three related, but different, light chains (VK1, JK2). The SP Fab fragments bind specifically to TPO with high affinities (6 x 10(-11)-2 x 10(-10) M) comparable to those of serum TPO autoantibodies. TPO autoantibodies represented by the SP Fab fragments are present in all 11 patients studied, constitute a high proportion (36-72%) of serum TPO autoantibodies in individual patients and interact with a conformational epitope on TPO.


Extensive posttranscriptional deletion of the coding sequences for part of nucleotide-binding fold 1 in respiratory epithelial mRNA transcripts of the cystic fibrosis transmembrane conductance regulator gene is not associated with the clinical manifestations of cystic fibrosis.

Cystic fibrosis (CF) is a recessive hereditary disorder, requiring both parental cystic fibrosis conductance transmembrane regulator (CFTR) genes to carry mutations for clinical disease to manifest, i.e., only 50% of normal CFTR gene expression is required to maintain a normal phenotype. To help define the minimum amount of normal CFTR gene expression necessary to maintain normalcy, we have capitalized on our prior observation (Chu, C.-S., B. C. Trapnell, J. J. Murtagh, Jr., J. Moss, W. Dalemans, S. Jallat, A. Mercenier, A. Pavirani, J.-P. Lecocq, G. R. Cutting, et al. 1991. EMBO [Eur. Mol. Biol. Organ] J. 10:1355-1363) that normal individuals can have up to 66% of bronchial CFTR mRNA transcripts that are missing exon 9, a region representing 21% of the sequence coding for the critical nucleotide (ATP)-binding fold 1 (NBF1) of the predicted CFTR protein. The study population included 78 individuals with no prior diagnosis of CF. Evaluation of bronchial epithelial cells (obtained by bronchoscopy) revealed that exon 9 was variably deleted in all individuals. Remarkably, there were four individuals, all greater than or equal to 35 yr, in whom bronchial epithelial cells exhibited 73, 89, 90, and 92% CFTR transcripts with inframe deletion of exon 9, respectively, despite normal sweat Cl- and no clinical manifestation of CF. In the context that only 8% or less of bronchial CFTR transcripts need exon 9 to maintain normal airway function, these observations strongly suggest that either exon 9 is not necessary for CFTR structure and/or function or that only a very small fraction of bronchial epithelial cells need to express normal CFTR mRNA transcripts with exon 9 to perform the function of CFTR sufficient to maintain a normal phenotype in vivo.


Differential pp60c-src activity in well and poorly differentiated human colon carcinomas and cell lines.

The results presented in this report demonstrate increased pp60c-src kinase activity associated with moderate to well differentiated colon tumors, corroborating previous observations by other groups. Extension of this analysis to include a small number of poorly differentiated colon carcinomas revealed src kinase activity comparable to that observed in normal colonic mucosa, considerably less than that observed in moderate/well differentiated lesions. Correlations of src kinase activity with differentiation was confirmed within a panel of colon cell lines where increased activity, associated with moderate/well differentiated lines, was accompanied by increased expression of pp60c-src protein. Use of an antiphosphotyrosine antibody in immunoprecipitation revealed the presence of novel phosphotyrosyl cellular substrates in human colon cell lines displaying elevated pp60c-src kinase activity. These observations suggest a role for the src protooncogene in colonic differentiation pathways.


Effects of interferon-gamma on nitric oxide synthase activity and endothelin-1 production by vascular endothelial cells.

Given the pivotal role suggested for IFN-gamma in immune diseases of the vascular wall, we investigated the effects of IFN-gamma on nitric oxide (NO) and endothelin-1 (ET-1) expression in bovine aortic endothelial cells (BAEC). We have previously reported that TNF-alpha enhanced NO synthase activity in BAEC as assessed by quantifying release of bioactive NO with reporter monolayers and measuring conversion of L-[14C]arginine to L-[14C] citrulline. In murine macrophages IFN-gamma synergizes with TNF-alpha or lipopolysaccharide to induce robust increases in calcium-independent NO synthase activity. In this study we have found that IFN-gamma alone failed to have a significant effect on NO synthase activity in BAEC. In contrast to murine macrophages, IFN-gamma inhibited TNF-alpha-stimulated induction of endothelial NO synthase activity in a concentration-dependent manner. This observation suggests that there is major difference in the response of BAEC and murine macrophages to IFN-gamma. A second major aim of this study was to determine the effect of IFN-gamma on preproET-1 mRNA expression and ET-1 secretion rates in BAEC. IFN-gamma alone had little or no effect on ET-1 mRNA levels and basal ET release when measured for 8 h. However, cotreatment with IFN-gamma potentiated the stimulatory effect of TNF-alpha on BAEC ET-1 mRNA transcript levels and ET release. In contrast, pretreatment of cells with IFN-gamma for 16-24 h blunted the stimulatory effect of TNF-alpha. These findings suggest that endothelial cell expression of vasoactive mediators is modified by the temporal interplay of at least two immune mediators, IFN-gamma and TNF-alpha.


Cultured human synovial fibroblasts rapidly metabolize kinins and neuropeptides.

Kinins and substance P have been implicated in the pathogenesis of inflammatory arthritis by virtue of their abilities to induce vasodilation, edema, and pain. The relative biological potencies of these peptides in vivo would depend at least in part upon their rates of catabolism in the joint. We hypothesized that human synovial lining cells may regulate intraarticular levels of kinins and neuropeptides via degradation by cell surface-associated peptidases. We exposed intact human synovial fibroblasts to kinins and substance P, in the presence or absence of specific peptidase inhibitors, and measured the amount of intact substrate remaining and degradation product(s) generated over time. Aminopeptidase M (AmM; EC 3.4.11.2), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) were identified on the cell surface of synovial cells. Bradykinin degradation was due entirely to NEP-24.11 (1.39 +/- 0.29 nmol/min per well). Lysylbradykinin was also degraded by NEP-24.11 (0.80 +/- 0.19 nmol/min per well); however, in the presence of phosphoramidon, AmM-mediated conversion to bradykinin (3.74 +/- 0.46 nmol/min per well) could be demonstrated. The combined actions of NEP-24.11 (0.93 +/- 0.15 nmol/min per well) and DAP IV (0.84 +/- 0.18 nmol/min per well) were responsible for the degradation of substance P. AmM (2.44 +/- 0.33 nmol/min per well) and NEP-24.11 (1.30 +/- 0.45 nmol/min per well) were responsible for the degradation of the opioid peptide, [Leu5]enkephalin. The identity of each of the three peptidases was confirmed via synthetic substrate hydrolysis, inhibition profile, and immunological identification. The profiles of peptidase enzymes identified in cells derived from rheumatoid and osteoarthritic joints were identical. These data demonstrate the human synovial fibroblast to be a rich source of three specific peptidases and suggest that it may play a prominent role in regulating peptide levels in the joint.


Proportion of glutamate- and aspartate-immunoreactive neurons in the efferent pathways of the rat visual cortex varies according to the target.

Immunohistochemistry, with antisera directed against glutamate (Glu) or aspartate (Asp), was combined with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) histochemistry to examine the distribution, morphology, and proportions of Glu- and Asp-containing neurons that give rise to corticofugal and callosal projections of the rat visual cortex. WGA-HRP injections in the dorsal lateral geniculate nucleus resulted in retrograde labelling of small and medium-sized cells throughout layer VI of the visual cortex. Of these cells, 60% were also Glu-immunoreactive and 61% Asp-positive. WGA-HRP injections in the superior colliculus labelled large and medium-sized neurons in the upper portion of layer V of the visual cortex. Of these cells, 46% were also stained for Glu and 66% for Asp. Injections in the pontine nuclei resulted in retrograde labelling of cells in the deeper part of cortical layer V. Retrogradely labelled cells, which were also immunoreactive for Glu or Asp, were large pyramidal cells. Corticopontine neurons, which were also Glu-positive, accounted for 42% of the total number of WGA-HRP labelled cells, whilst for Asp-positive neurons this percentage was 51%. Finally, after injections in the visual cortex, retrogradely labelled small and medium-sized cells were found throughout layers II-VI in the contralateral visual cortex. Of these neurons, 38% were also labelled for Glu while 49% were also Asp-immunoreactive. The present results demonstrate that substantial proportions of projection neurons in the rat visual cortex are immunoreactive for Glu or Asp, suggesting that these excitatory amino acids are the major transmitters used by the cortical efferent systems examined. Furthermore, the proportions of these immunoreactive neurons in the efferent pathways vary according to the target.


Noradrenergic innervation of the thalamic reticular nucleus: a light and electron microscopic immunohistochemical study in rats.

Fluoro-ruby injections in the rat locus coeruleus result in scattered chain-like arrays of varicose anterogradely labeled axons within the thalamic reticular nucleus of rats. An abundant meshwork of axons giving rise to en passant boutons is detected immunohistochemically within this thalamic nucleus by means of an antibody to dopamine-beta-hydroxylase (DBH). The density of DBH-positive axonal boutons within the reticular nucleus neuropil is greater than that found in the relay nuclei of the dorsal thalamus (with the exception of the anterior group nuclei). Single DBH-positive axons appear to contact both proximal and distal dendrites and occasionally the somata of reticular nucleus neurons. Labeled axons are seen closely juxtaposed not only to the swollen segments of the beaded reticular neuron dendrites, but to the constricted segments as well. Electron microscopic examination of DBH-positive axon terminals within the reticular nucleus neuropil indicates that many of the axonal boutons detected light microscopically participate in asymmetric synaptic contacts. The postsynaptic densities of these synapses are thicker than those of nearby symmetric synapses, but often subtend a shorter length of the postsynaptic membrane than the densities associated with other nearby asymmetric synapses. These observations indicate that the ascending noradrenergic system, in addition to influencing the dorsal thalamus and the cerebral cortex directly, is well situated to influence signal transmission through the nuclei of the dorsal thalamus indirectly via a moderately dense terminal projection upon the thalamic reticular nucleus.