Efficacy of moclobemide in different patient groups: a meta-analysis of studies. Whilst tricyclic antidepressants are efficacious in all depressive syndromes, classical MAO-inhibitors differ substantially from them in their action. They are considered less effective in general and not very effective in endogenous depression, but recommended for the treatment of 'atypical' depression. A new class of RIMA (Reversible Inhibitors of MAO-A) represented by moclobemide requires a change in clinical thinking on antidepressants. Moclobemide shows the same efficacy in depression as tricyclics: its effects are similar in unipolar and bipolar affective disorders, and in patients with major depressive episode superimposed on dysthymia (double depression). As with classical antidepressants, the response rate tends to be lower, but is still present in psychotic depression. Agitated depressives do not respond less well than non-agitated patients to moclobemide. Patients meeting DSM-III-R criteria for major depression with melancholia tend to respond better than non-melancholics, but this may be associated with the significantly higher baseline severity observed in melancholics. A slightly higher response rate in patients without concomitant benzodiazepine treatment, compared to those with benzodiazepine comedication, may also be related to baseline differences in the severity of depression. Elderly depressives respond less well than younger patients to classical antidepressants, but with moclobemide, elderly patients do as well as younger ones.


Biochemical effects of high single doses of moclobemide in man: correlation with plasma concentrations. The effects of high single doses of moclobemide (300, 450 and 600 mg given at the end of a standardized meal) on plasma levels of several catecholamines and their deaminated metabolites, and on plasma levels of pituitary hormones were determined in eight healthy young male volunteers in a randomized, double-blind, placebo-controlled study. Assessment of the i.v. tyramine potentiation and determination of the plasma levels of moclobemide were also performed. The tyramine sensitivity factor at 2 h after dosing was about 2.1, with no significant differences between the doses used. The inhibitory activity of moclobemide on MAO-A was reflected in significant reductions of plasma concentrations of DHPG and 5-HIAA. No clear differences were detected between the moclobemide doses. Prolactin plasma concentrations were only slightly increased after the two higher doses. The plasma concentrations of moclobemide were very much in agreement with those found in previous studies under similar experimental conditions. Thus, single oral doses of 300, 450 and 600 mg moclobemide demonstrated marked inhibition of MAO-A activity, whereas a single dose of 300 mg induced a near-maximum effect.


Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1. An indefinite survival of cardiac allografts between fully incompatible mice strains was observed when monoclonal antibodies (MAbs) to intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) were simultaneously administered after the transplantation for 6 days. Mice with long-term surviving cardiac allografts accepted skin grafts from the donor-strain but rejected skin grafts from a third-party strain. Because MAbs to ICAM-1 or LFA-1 alone were insufficient for prolonged tolerance, the two MAbs probably acted synergistically to induce specific unresponsiveness. Thus, ICAM-1----LFA-1 adhesion participates in the induction of allograft rejection and MAbs may be useful as therapeutic agents.


Barrett's esophagus: observations on diagnosis and management. Barrett's esophagus is a premalignant condition that may often pass unrecognized in clinical practice. In adults, this condition is generally believed to be caused by chronic gastroesophageal reflux resulting in a metaplastic change in the epithelium of the esophagus. Diagnosis of Barrett's esophagus is established by careful biopsy of the involved esophageal mucosa. Periodic surveillance is recommended because of the risk of carcinoma. Antireflux surgery has not been shown to result consistently in the regression of the metaplastic epithelium, but potent acid suppression offers a new therapeutic approach that leads to healing of esophagitis and the potential regression of Barrett's epithelium.


A single base change at a splice acceptor site leads to a truncated CAD protein in Urd-A mutant Chinese hamster ovary cells. We have previously reported the isolation and characterization of mutant Chinese hamster ovary (CHO-K1) cells of the Urd-A complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo UMP biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein CAD, which contains these three enzyme activities. We report here that a single base change of G to A at a highly conserved RNA splice acceptor site is responsible for the phenotype of this mutant. In addition to a small amount of apparently normal CAD mRNA, this mutation causes production of two alternative forms of CAD mRNA in the mutant, one that includes the intron just prior to the mutation and one that excludes the exon just after the mutation. The affected splice site is located at the intron-exon boundary just preceding the exon that encodes the beginning of the aspartate transcarbamylase (ATCase) domain of the CAD protein. Both intron inclusion and exon exclusion during RNA processing introduce a translation stop codon upstream of the region encoding this domain, resulting in the production of the truncated CAD protein seen in the Urd-A mutant. This mutation also results in markedly decreased levels of CAD mRNA and protein in the mutant.


Subdural empyema complicating bacterial meningitis in a child: enhancement of membranes with gadolinium on magnetic resonance imaging in a patient without enhancement on computed tomography. Subdural empyema is a known yet infrequent complication of bacterial meningitis. Subdural effusions occur frequently with meningitis in children and usually resolve spontaneously or with subdural taps. Subdural empyema should be suspected when a patient fails to respond to antibiotic therapy or worsens neurologically. Computed tomography (CT) scans with contrast often show enhancement of subdural collections when an empyema exists. However, this is not true all of the time. We present a case of subdural empyema complicating bacterial meningitis in a 4 month old in which CT enhancement was not present yet magnetic resonance imaging (MRI) scans with gadolinium demonstrated intense enhancement. For comparison, we present a second case of a child with sterile subdural effusions due to meningitis that demonstrates an absence of contrast enhancement on MRI studies. MRI scans with contrast may offer a more sensitive means of making an early diagnosis of subdural empyema.


Changes in mononuclear peripheral blood cells in cattle with foot-and-mouth disease. The percentages and absolute numbers of mononuclear peripheral blood cells (MNC) were studied in vaccinated (Vac) and non-vaccinated (control) cattle, challenged with foot-and-mouth disease virus (FMDV). All Vac cattle but none of the controls resisted challenge. Cell populations were studied immediately before and one week after challenge, by direct and indirect immunofluorescence, using polyclonal and monoclonal antibodies against different bovine markers. Total B-lymphocytes, as assessed with polyclonal anti-IgG (H + L) antisera, as well as total mononuclear cells, were normal before and after infection, and did not change in Vac or control groups. Before challenge Vac cattle had higher numbers of ILA-29+ (a putative marker for null cells or, alternatively, for gamma delta T-cells) than control cattle. After challenge, in control cattle, the number of total T-cells, BoT4-bearing (helper) T-cells and BoT8-bearing (cytotoxic/suppressor) T-cells were decreased, while IgM-bearing B-lymphocytes, as well as monocyte/macrophage cells were increased. The number of IL-A29-bearing T-cells did not change after infection in either group. After challenge, Vac cattle also showed increased numbers of IgM-bearing B-lymphocytes and monocyte/macrophage cells, whereas T-subpopulations did not change significantly.


Kinetics of HIV-1 interactions with sCD4 and CD4+ cells: implications for inhibition of virus infection and initial steps of virus entry into cells. The mechanisms of human immunodeficiency virus (HIV-1) entry into CD4+ cells and HIV-1 inactivation by sCD4 were studied by analyzing the kinetics of inhibition of viral infection by sCD4 and the kinetics of fusion of CD4+ cells with intact virions labeled with the lipid fluorophore octadecylrhodamine (R18). sCD4 inhibited HIV-1 infection much more effectively when preincubated with virus prior to interaction with CD4+ cells than when mixed simultaneously with virions and cells. The kinetics of inhibition of infection was much slower at 4 degrees and at low sCD4 concentrations than at 37 degrees and at high sCD4 concentrations. In the absence of sCD4, attachment of virus to cells leading to productive infection occurred within 10-30 min. Fusion of the virions with cells started after a 1-2 min lag time and was complete within 15 min. In high-density cell suspensions (5 x 10(7) cells/ml), even very high sCD4 concentrations (100 micrograms/ml) failed to block viral infection during simultaneous mixing of cells, sCD4 and HIV-1. We conclude that the kinetics of sCD4-virus interaction and the competition of sCD4 with the cell surface associated CD4 for the virus are crucial factors in the inhibition of HIV-1 infection by sCD4. These results provide insight into mechanisms of viral penetration into cells and should be considered when designing new approaches for AIDS therapy.


Mechanisms of tetrazepam action on spasticity. This investigation assessed the mechanisms of Tetrazepam action on spasticity using a battery of electromyographic methods. Thirty patients with post-stroke spastic hemiparesis treated with Tetrazepam took part in the investigation. A questionnaire for assessment of subjective improvement after treatment used a 5-point scale. The 5-point scales were used to assess muscle tone, muscle strength and tendon reflexes. A battery of electromyographic methods was used to analyse different mechanisms of spasticity: for alpha-motoneuron activity--the F-wave parameters; for gamma-motoneuron activity--the TA/H amplitude ratio; for presynaptic inhibition--the ratio of H-reflex maximal amplitudes before and after vibration on the Achilles tendon (Hvibr/Hmax); for common interneuron activity--the flexor reflex parameters. Our results revealed that Tetrazepam reduces tone in spastic muscles and has a slight effect on tendon hyperreflexia. It has no influence on muscle strength, Babinski sign and ankle clonus. Tetrazepam acts by decreasing motoneurone activity and increasing presynaptic inhibition.


Immune response to a murine coronavirus: identification of a homing receptor-negative CD4+ T cell subset that responds to viral glycoproteins. The lymphocyte proliferative response to mouse hepatitis virus, strain JHM (MHV-JHM), a well-described cause of chronic and acute neurological infections, has been studied using vaccinia virus recombinants expressing individual MHV proteins. The surface (S) and transmembrane (M) glycoproteins were the most active proteins in causing proliferation of lymphocytes isolated from immunized adult mice, whereas lymphocytes from persistently infected mice proliferated only in response to the S protein. The cells from immunized mice which proliferated most actively in response to MHV were positive for the CD4 antigen and secreted interferon-gamma. In addition, the most responsive subset of cells did not express gp90MEL-14, the lymph node-specific homing receptor. The results identify a subpopulation of CD4+ T cells that may be an important component of the cell-mediated immune response to this virus. The data also suggest that response to the M protein is important in preventing disease progression in C57BL/6 mice since cells which recognize this protein are absent from persistently infected mice.


Effects of antipsychotic drugs on memory functions of schizophrenic patients. In this research we investigated the effects of 4 antipsychotic drugs with different anticholinergic components on different memory functions of schizophrenic patients. Drugs were administered in cumulative doses and memory was tested 90 min after each drug was administered. The results show that chlorpromazine and thioridazine impaired short-term verbal memory after 6 h of sequential administration. Trifluoperazine and haloperidol improved short-term verbal memory from the third to the fifth administration. Immediate memory, long-term memory and visual short-term memory were not impaired by any drug.


Immunologic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Expression of proteins associated with immune function was investigated immunohistochemically in postmortem brain and spinal cord of patients with amyotrophic lateral sclerosis (ALS). Reactive microglia/macrophages displaying high levels of leukocyte common antigen (LCA), the immunoglobulin receptor Fc gamma R1, lymphocyte function associated molecule-1 (LFA-1), the complement receptors CR3 and CR4, the class II major histocompatibility complex molecules HLA-DR, HLA-DP and HLA-DQ and common determinants of the class I HLA-A,B,C complex were abundant in affected areas in ALS. These areas included the primary motor cortex, motor nuclei of the brain stem, the anterior horn of the spinal cord, and the full extent of the corticospinal tract. A significant number of T lymphocytes of the helper/inducer (CD4+) and cytotoxic/suppressor (CD8+) subtypes were observed marginating along the walls of capillaries and venules and extending into the parenchyma of affected areas. Clusters of complement activated oligodendroglia as well as degenerating neurites positive for C3d and C4d were frequently detected in ALS-affected areas. These data provide evidence of immune-effector changes in ALS. They are consistent with an autoimmune or slow virus theory of the disorder, but may reflect only secondary changes.


Characterization of the endocrine cells in the pancreatic-bile duct system of the rat. Six types of endocrine cells showing immunolabelling against gut or pancreatic islet hormones were identified in the pancreatic-bile duct system of the normal adult rat at the light and electron microscopic levels. They were located within the epithelial lining of the duct system from the intercalated portion to its duodenal opening. However, the distribution and frequency of each endocrine cell varied along the length of the duct system. While insulin, glucagon, somatostatin, and pancreatic polypeptide cells were widely distributed along the entire duct system, small numbers of cholecystokinin and serotonin cells were confined to the terminal portion. A considerable number of somatostatin cells were concentrated in gland-like pouches of the terminal portion of the common pancreatic-bile duct. When the accessory pancreatic duct was present, insulin, glucagon, and somatostatin cells were also found in its epithelial lining. Electron microscopically, the specific content of the secretory granules of all endocrine cells was confirmed by immunolabelling or cytochemical staining. Further the characteristics of the secretory granules of each endocrine cell type corresponded to those present in the same kind of endocrine cells in gut or pancreatic islet. The duct endocrine cells displayed a particular ultrastructural appearance. The "open type cells" were highly polarized, with their apical cytoplasmic process reaching the duct lumen, whereas "closed type cells" showed long basal cytoplasmic processes with no connection with the duct lumen. In general, insulin, and somatostatin cells were of the "open type", while no morphological connection with the duct lumen was found for glucagon and pancreatic polypeptide cells. The presence of various duct endocrine cells with their particular ultrastructural appearance implies that they may take part in modulating the function of the duct system.


Cetirizine does not influence the immune response. Antihistamines are frequently employed in the treatment of allergic rhinitis and urticaria-angioedema syndrome. We analyzed the in vitro effects of cetirizine on the immune response. To this end the proliferation of peripheral mononuclear cells induced by mitogen and by -CD3, -CD2, or -CD28 monoclonal antibodies has been studied. Since the plasma peak of cetirizine following ingestion of 10 mg is about 1 microgram/mL, the drug was tested in the cultures at the concentration of 0.1, 1, or 10 micrograms/mL. No influence of cetirizine on T cell proliferation was detected. We also evaluated the effect of cetirizine on the expression of the following markers expressed by T cells upon activation: lymphocyte markers ICAM-1, HLA-DR, and CD25 surface expression, alpha-1-acid glycoprotein has been also studied. There was no effect of cetirizine on the investigated immunologic parameters; these data acquire clinical relevance when related to previous reports showing a depression of the immunologic response exerted by other compounds such as ketotifen and theophylline and when related to the recent data about the modulation of ICAM-1 expression on eosinophils by cetirizine. Cetirizine does not affect ICAM-1 expression of lymphocyte membrane.


Level of blood pressure and risk of myocardial infarction among treated hypertensive patients. To examine the association between the level of treated blood pressure and the incidence of myocardial infarction, we conducted a population-based case-control study of 912 members of a health maintenance organization who were receiving standard clinical treatment for hypertension. We found a J-shaped relationship between the most recently measured diastolic blood pressure and the risk of myocardial infarction, the lowest risk occurring at 84 mm Hg. The relative risk of myocardial infarction at 60 mm Hg was 2.07 (95% confidence interval, 0.86 to 5.01), and at 100 mm Hg, 1.45 (95% confidence interval, 1.02 to 2.06). Treated systolic pressure bore a linear relationship to the risk of infarction. We conclude that the optimum target range for diastolic blood pressure in hypertensive patients may be 84 to 90 mm Hg. Levels outside this range may be associated with increased risk of myocardial infarction.


Aerobic fitness and hormonal responses to prolonged sleep deprivation and sustained mental work. This study examined the influence of aerobic fitness on the responses of selected hormones to the combined stressors of sleep deprivation (SD) and sustained mental work. Six aerobically high fit (HF) (VO2max greater than 50 ml.kg-1.min-1) and six average fit (AF) (VO2max less than 40 ml.kg-1.min-1) female subjects were subjected to a period of sleep loss of 60 h during which time they performed sustained mental tasks with no physical activity component. Venous blood samples were drawn every 12 h at 1330 hours and 0130 hours and plasmas analyzed for cortisol, growth hormone (hGH), prolactin, thyroxine (T4), triiodothyronine (T3), and reverse-triiodothyronine (rT3). For cortisol, both the HF and AF groups exhibited the normal high-daytime and low-nighttime pattern of secretion, with levels increasing significantly as the duration of SD increased. The normal elevations of hGH and prolactin levels during normal sleep were suppressed during SD. No significant fitness effects were found for cortisol, hGH, and prolactin responses. Plasma levels of T4, T3, and rT3 increased significantly during SD, with highly fit subjects exhibiting higher levels of these hormones than those of average fitness. We suggest that aerobic fitness may influence the peripheral metabolism of T4 during SD, but that aerobic fitness does not influence the regulation of the classical stress hormones during SD.


Effect of L-glutamate on 2-oxoglutarate decarboxylase in Euglena gracilis. The effect of tricarboxylic acid-cycle intermediates and related compounds on 2-oxoglutarate decarboxylase activity was investigated. The addition of L-glutamate to Euglena cells grown on glucose/(NH4)2SO4 medium resulted in an increase in 2-oxoglutarate decarboxylase activity, which was abolished by the simultaneous addition of cycloheximide. Immunochemical titration, immunoblot analysis and labelling in vivo with antibody raised against 2-oxoglutarate decarboxylase showed that the increase in 2-oxoglutarate decarboxylase activity was due to synthesis of new protein and not to activation of pre-existing protein. The experimental results reported here demonstrate that L-glutamate is assimilated by the pathway, via 2-oxoglutarate, that consists of L-glutamate-oxaloacetate aminotransferase, 2-oxoglutarate decarboxylase and succinate semialdehyde dehydrogenase, rather than by the gamma-aminobutyrate shunt, consisting of L-glutamate decarboxylase and gamma-aminobutyrate aminotransferase.


A structural motif that defines the ATP-regulatory module of guanylate cyclase in atrial natriuretic factor signalling. Atrial natriuretic factor (ANF)-dependent guanylate cyclase is a single-chain transmembrane-spanning protein, containing an ANF receptor and having catalytic activity. ANF binding to the receptor domain activates the catalytic domain, generating the second messenger cyclic GMP. Obligatory in this activation process is an intervening step regulated by ATP, but its mechanism is not known. Through a programme of site-directed and deletion mutagenesis/expression studies, we report herein the identity of a structural motif (Gly503-Arg-Gly-Ser-Asn-Tyr-Gly509) that binds ATP and amplifies the ANF-dependent cyclase activity; this, therefore, represents an ATP-regulatory module (ARM) of the enzyme, which plays a pivotal role in ANF signalling.


High blood pressure: hunting the genes.

High blood pressure: hunting the genes. High blood pressure is a disease of unknown cause. Family history of the disease indicates higher risk, but it is not known which genes are involved or how they interact with environmental influences to produce the disorder. Molecular biology offers an approach to problems that have not so far been solved by classical physiology or biochemistry. By analysing polymorphic variation in chromosome markers such as minisatellite sequences, or by restriction fragment polymorphism analysis of candidate genes, attempts are being made to link genetic variations with hypertension. In genetically hypertensive rats, hypertension is associated with a polymorphism of the renin gene and with other loci on chromosomes 10 and 18. The role of these loci in human hypertension remains to be determined. Other genes such as sodium-lithium countertransport may be involved. Environmental factors such as stress or salt intake could influence the rate or timing of expression of certain genes and thus result in hypertension.


Differential effects of vecuronium on diaphragm and geniohyoid muscle in anaesthetized dogs. We have examined the sensitivity of the geniohyoid, an upper airway dilating muscle, to vecuronium in 12 anaesthetized dogs undergoing mechanical ventilation of the lungs and compared it with that of the diaphragm. Dogs were allocated randomly to two groups: pentobarbitone alone (group 1, n = 7); pentobarbitone combined with 0.2 MAC (0.44%) of enflurane anaesthesia (group 2, n = 5). Supramaximal single twitch stimulations (0.1 Hz) were applied to the phrenic nerves in the upper thorax and the geniohyoid branches of the hypoglossal nerves at the neck. The evoked responses were assessed by the transdiaphragmatic pressure (Pdi) and the isometric force of the geniohyoid muscles (Tgh) until complete recovery of these variables after i.v. administration of vecuronium 0.02 mg kg-1. In both groups, the magnitude of the depression of twitch response was greater and time required to reach control amplitude was longer in the geniohyoid than the diaphragm. The depression of Tgh was significantly greater in group 2 than in group 1, whereas no change was observed in Pdi between the two groups. We conclude that the geniohyoid is more sensitive to vecuronium than the diaphragm and the differential effects of vecuronium are facilitated by a low concentration of enflurane.


Propofol potentiates both pre- and postsynaptic effects of vecuronium in the rat hemidiaphragm. We have measured twitch tension in response to train-of-four stimulation in rat isolated phrenic nerve-hemidiaphragm preparations. Propofol inhibited nerve evoked twitch tension, with 50% inhibition occurring at 420 (SD 29) mumol litre-1. Although propofol 100 mumol litre-1 by itself had no effect on nerve evoked twitch tension, it potentiated the neuromuscular blocking effects of vecuronium. The decrease in train-of-four ratio with vecuronium was directly proportional to the degree of twitch inhibition, regardless of whether twitch was depressed by vecuronium alone or in combination with propofol. The finding that the train-of-four ratio was a function of the degree of block, rather than simply a function of vecuronium concentration, indicates that propofol also contributed to train-of-four fade and potentiated both pre- and postsynaptic effects of the neuromuscular blocker. The concentrations of propofol used in this study are much greater than human therapeutic blood concentrations, which are typically 25-35 mumol litre-1 (4-6 micrograms ml-1) immediately after a bolus dose of 2 mg kg-1, suggesting that neither muscle weakness nor potentiation of vecuronium-induced neuromuscular block should be of concern at propofol concentrations occurring clinically.


Tumor necrosis factor and chemotherapeutic drugs targeted at DNA topoisomerase II for the treatment of genitourinary malignancies. Recombinant TNF as a single agent for human cancer appears to be of limited value. However, rTNF has synergistic anticancer effects when combined with chemotherapeutic drugs targeted at DNA topoisomerase II. This effect of rTNF has been observed in several in vitro and in vivo tumor models, both in animal and human studies. The mechanism of this interaction appears to involve lesions to the DNA of tumor cells mediated by inhibition of DNA topoisomerase II. The combinations of rTNF plus doxorubicin and rTNF plus etoposide administered systemically are currently under evaluation by clinical trials in patients with advanced cancers. Determination of the efficacy of such combination therapy must await the completion of phase I and II trails. Other routes of administration that might increase the local concentration of rTNF and could be combined with topoisomerase II-targeted drugs include intravesical administration and the use of tumor-infiltrating lymphocytes.


Cholinergic terminals in the cat visual cortex: ultrastructural basis for interaction with glutamate-immunoreactive neurons and other cells. Acetylcholine (ACh) is one of the transmitters utilized by extra-thalamic afferents to modulate stimulus-driven neurotransmission and experience-dependent plasticity in the visual cortex. Since these processes also depend on the activation of glutamatergic receptors, cholinergic terminals may exert their effects via direct modulation of excitatory neurotransmission. The objective of this study was to determine whether the ultrastructural relationships between cholinergic terminals, glutamate-immunoreactive neurons, and other unlabeled cells support this idea. Sections from aldehyde-fixed visual cortex (area 17) of adult cats were immunolabeled for the following molecules: (1) choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme; (2) L-glutamate; or (3) ChAT simultaneously with L-glutamate by combining electron-microscopic immunogold and immunoperoxidase techniques. None of the cortical terminals were dually labeled, suggesting that (1) the labeling procedure was free of chemical or immunological cross reactions; and (2) glutamate immunoreactivity probably reflects the transmitter, and not metabolic, pool of L-glutamate. Comparisons between cholinergic and noncholinergic axons revealed that (1) ChAT-immunoreactive axons formed fewer identifiable synaptic contacts within single ultrathin sections (P less than 0.01 using chi-square test); and (2) more of the cholinergic axons occurred directly opposed to other terminals (P less than 0.0015 by chi-square test), including 21% of which resided directly across asymmetric, axo-spinous junctions. Dual labeling showed that a third of the synaptic targets for cholinergic terminals contained detectable levels of glutamate immunoreactivity. Some of the axo-spinous junctions juxtaposed to cholinergic axons also exhibited glutamate immunoreactivity presynaptically. These observations provide ultrastructural evidence for direct, cholinergic modulation of glutamatergic pyramidal neurons within the mammalian neocortex. Prevalence of juxtapositions between cholinergic terminals and axo-spinous synapses supports the following ideas: (1) ACh may modulate the release of noncholinergic transmitters, including Glu; (2) Glu may modulate ACh release; and (3) these processes may be concurrent with cholinergic modulation of glutamatergic synapses at postsynaptic sites.


Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis. The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630----Glu or Tyr632----Phe caused no effect on the enzyme activity, that of Tyr632----Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.


Are proton symports in yeast directly linked to H(+)-ATPase acidification? Transport of amino acids in Saccharomyces cerevisiae is an H(+)-driven secondary active transport. Inhibitors of the plasma membrane H(+)-ATPase, particularly heavy water, diethylstilbestrol and suloctidil, were shown to affect the H(+)-extruding ATPase activity as well as the ATP-hydrolyzing activity, to a similar degree as they inhibited the transport of amino acids. The inhibitors had virtually no effect on the membrane electric potential or on the delta pH which constitute the thermodynamically relevant source of energy for these transports. Transport of acidic amino acids was affected much more than that of the neutral and especially of the basic ones. The effects were greater with higher amino acid concentrations. All this is taken as evidence that the amino acid carriers respond kinetically to the presence of protons directly at the membrane site where they are extruded by the H(+)-ATPase, rather than to the overall protonmotive force.


Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions. We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.


Lack of association between an RFLP near the D2 dopamine receptor gene and severe alcoholism. Blum et al (1990) have recently examined a restriction fragment length polymorphism (RFLP) detected by TaqI RFLP to the dopamine D2 receptor gene (DRD2) in deceased alcoholics and nonalcoholics, and reported an association between alcoholism and the A1 allele. Subsequent studies, however, by other investigators have failed to confirm this. We have examined the DRD2 TaqI RFLP in 47 living Caucasian males with severe alcoholism. All alcoholic subjects were thoroughly characterized by a structured interview, and met DSM-III-R criteria for alcohol dependence. Only 9/47 (19%) (1990) of these alcoholics had the AI allele compared to 14/22 (64%) reported by Blum et al. This rate was not significantly different from the rates reported in control populations by Blum et al (1990), CEPH, or Bolos et al (1990), and differed only slightly from those reported by Grandy et al (1990). Alcoholics selected for severe medical complications also displayed a similar rate. Our data do not support an association between alcoholism and the D2 dopamine receptor gene in this population.


Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations. This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.


Absolute requirement of CD11/CD18 adhesion molecules, FcRII and the phosphatidylinositol-linked FcRIII for monoclonal antibody-mediated neutrophil antihuman tumor cytotoxicity. We have previously shown that 3F8, a murine IgG3, monoclonal antibody (MoAb) specific for the ganglioside GD2, mediates tumor cell kill in vitro and in vivo. We now describe receptor requirements of polymorphonuclear leukocytes (PMN) in 3F8-mediated cytotoxicity (ADCC) of human GD2 (+) melanoma and neuroblastoma cell lines. PMN from a child with leukocyte adhesion deficiency (LAD) were devoid of CD11/CD18 adhesion molecules and mounted no detectable ADCC. MoAb to CD11b, CD11c, and CD18 each efficiently blocked ADCC by normal PMN. In contrast, a panel of different MoAbs to CD11a had no significant inhibitory effect on ADCC, a finding consistent with the low-to-absent expression of the CD11a ligand, intercellular adhesion molecule-1, on the target cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the expression of CD11b, CD11c, and CD18 on normal PMN, decreased the expression of Fc receptors (FcR), and enhanced ADCC by normal but not by LAD PMN. MoAbs to FcRII and FcRIII each efficiently blocked ADCC; anti-FcRI MoAb had no effect. Flow cytometry using anti-FcRII MoAb versus anti-FcRIII MoAb did not show cross competition, suggesting that inhibition of ADCC was not a steric effect resulting from FcRII proximity to FcRIII. PMN deficient in FcRIII (obtained from patients with paroxysmal nocturnal hemoglobinuria) and PMN depleted of FcRIII by treatment with elastase or phosphatidylinositol (PI)-specific phospholipase C produced low ADCC, supporting a role for the PI-liked FcRIII. Thus, optimal ADCC using human PMN, human solid tumor cells, and a clinically active MoAb (conditions that contrast with the heterologous antibodies and nonhuman or nonneoplastic targets used in most models of PMN ADCC) required CD11b, CD11c, FcRII, and the PI-linked FcRIII. Furthermore, in this clinically relevant system, GM-CSF enhancement of antitumor PMN ADCC correlated with increased expression of CD11/CD18 molecules.


High expression of the mdr3 multidrug-resistance gene in advanced-stage chronic lymphocytic leukemia. Chronic lymphocytic leukemia (CLL) is characterized by an often indolent course with a poor therapeutic response at advanced stage. We investigated the expression of the human multidrug resistance genes mdr1 and mdr3 in 31 patients with CLL. Using specific probes for mdr1 and mdr3 mRNA, respectively, expression of both genes could be found in 29 of 31 patients. Of those, nine had high expression of mdr1 and 13 of mdr3. Although 29 of 31 patients showed coexpression of mdr1 and mdr3, the mRNA levels were not interrelated. Prior treatment did not significantly influence the level of mdr1 or mdr3 expression. In patients with advanced CLL (Rai stage 3 + 4) the mdr3 expression was significantly higher than in early-stage CLL (Rai stage 0 to 2) (mean +/- SEM, 25.4 +/- 4.2 U v 4.2 +/- 1.1 U; P less than .0001). Such a difference was not present for mdr1 expression (21.5 +/- 4.3 U v 10.7 +/- 3.1 U; P = .09). These data indicate that advanced-stage CLL is associated with an increased mdr3 expression, which may concur with a decreased sensitivity to chemotherapy.


Chromosomal loss and deletion are the most common mechanisms for loss of heterozygosity from chromosomes 5 and 7 in malignant myeloid disorders. We have examined a population of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) for loss of heterozygosity of polymorphic markers on chromosomes 5 and 7. The rationale for this study was the observation that the majority of patients with therapy-related leukemia (t-AML or t-MDS), resulting from cytotoxic treatment for prior malignancies, have loss of chromosome 5 and/or 7 or deletions involving the long arms of one or both of these chromosomes. This cytogenetic finding suggested that tumor-suppressor genes, important in the development of AML, may be located in these chromosomal regions. We analyzed a total of 60 patients, 43 with primary MDS/AML de novo and 17 with t-MDS/t-AML. Leukemia cells were evaluated for restriction fragment length polymorphisms (RFLPs). Leukemia cell genotypes were compared with lymphoblastoid cell genotypes from the same patients. Two cases of loss of heterozygosity were identified from chromosomes lacking visible deletions: one involving chromosome 5 in a patient with AML de novo who had a visible deletion of 5q at a later stage of the disease, and one involving chromosome 7 in a patient with t-AML. We conclude that allele loss from loci on chromosomes 5 and 7 in MDS/AML, when it occurs, usually results from major deletion or simple chromosome loss, rather than from mitotic recombination or chromosome loss with duplication of the remaining homologue.


CD11/CD18-independent neutrophil adherence to laminin is mediated by the integrin VLA-6. Regulated adherence of polymorphonuclear leukocytes (PMNs) to endothelium and subendothelial matrix is a critical event for PMN localization at and migration into inflammatory sites. We previously reported that human PMNs stimulated in vitro adhere to laminin, the major glycoprotein of mammalian basement membrane, by both CD11/CD18 (beta 2 integrin)-dependent and CD11/CD18-independent mechanisms. This CD11/CD18-independent adherence is inhibited by monoclonal antibodies (MoAbs) directed against the beta 1 subunit of integrins (very late antigens [VLA]). The specific PMN VLA receptor responsible for stimulated CD11/CD18-independent PMN adherence to laminin was not elucidated. We show here that this CD11/CD18-independent adherence is mediated by a member of the beta 1 integrins, VLA-6. MoAbs GoH3 and 450-30, which bind the alpha 6 subunit of VLA-6, significantly reduced adherence of phorbol myristate acetate-stimulated PMNs to laminin-coated surfaces when CD11/CD18-independent adherence was blocked with anti-CD11/CD18 MoAbs. Furthermore, GoH3 completely inhibited stimulated adherence of CD11/CD18-deficient PMNs to laminin. Analysis by flow cytometry showed that human PMNs express VLA-6. The PMN alpha 6 is identical in size and pl to the platelet alpha 6, but the PMN beta 1 exhibits considerable heterogeneity in molecular weight compared with the platelet beta 1. This activation-dependent adherence receptor for laminin may play a role in PMN interaction with basement membrane laminin during PMN movement through vascular walls.


Management of elderly patients with sustained hypertension. To assess the clinical benefits of treating hypertension in elderly patients and to derive practical guidelines regarding indications, goals, and forms of treatment. Review of six published randomised trials. Active treatment of hypertension in elderly patients was associated with significant improvements in several indices of cardiovascular morbidity and mortality, particularly the incidence of fatal and non-fatal strokes. On the basis of the trial data, combined systolic and diastolic hypertension was defined as a sustained systolic pressure greater than 160 mmHg and diastolic pressure greater than 90 mmHg. There is convincing evidence that efforts should be made to reduce both systolic and diastolic pressures to below these levels in patients up to the age of 80 years. Isolated systolic hypertension was defined as a systolic pressure greater than 160 mmHg in the presence of a diastolic pressure less than 90 mmHg. Two trials reported benefit from the treatment of isolated systolic hypertension in patients up to the age of 80, and further trials are underway to support or refute this recommendation. Diuretics have an established role in the management of hypertension in elderly patients; beta adrenoceptor antagonists have given variable results, and the benefits are less impressive than with diuretic based regimens. Newer agents show promise in the treatment of elderly patients, particularly in the presence of coexisting disease, but their effects on morbidity and mortality have not been evaluated in large randomised trials. Diuretics rather than beta blockers are the treatment of choice for patients with uncomplicated hypertension, but combinations of drugs may be required in as many as 50% of patients.


Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cytochrome b2 reaches the intermembrane space of mitochondria by transport into the matrix followed by export across the inner membrane. While in the matrix, the protein interacts with hsp60, which arrests its folding prior to export. The bacterial-type export sequence in pre-cytochrome b2 functions by inhibiting the ATP-dependent release of the protein from hsp60. Release for export apparently requires, in addition to ATP, the interaction of the signal sequence with a component of the export machinery in the inner membrane. Export can occur before import is complete provided that a critical length of the polypeptide chain has been translocated into the matrix. Thus, hsp60 combines two activities: catalysis of folding of proteins destined for the matrix, and maintaining proteins in an unfolded state to facilitate their channeling between the machineries for import and export across the inner membrane. Anti-folding signals such as the hydrophobic export sequence in cytochrome b2 may act as switches between these two activities.


Cyproterone acetate induces DNA damage in cultured rat hepatocytes and preferentially stimulates DNA synthesis in gamma-glutamyltranspeptidase-positive cells. The synthetic anti-androgen and progestin cyproterone acetate (CPA) is known to increase the liver tumor rate in rats. The tumorigenicity of CPA has been attributed to a tumor-promoting activity of the steroid. In order to discover whether CPA acts directly on preneoplastic liver cells or via indirect effects, we investigated whether CPA stimulates replicative DNA synthesis in vitro in hepatocytes isolated from carcinogen-treated rats (two-thirds hepatectomy, 1 x 30 mg diethylnitrosamine per kg and 0.1% phenobarbital in the drinking water) and whether the degree of stimulation differs in gamma-glutamyltranspeptidase (GGT)-positive, putatively preneoplastic and GGT-negative, 'normal' hepatocytes. The possibility that CPA might also have initiating potential was investigated by studying its effects on DNA repair synthesis. Stimulation of [3H]thymidine incorporation into the DNA by CPA was only observed in the presence of epidermal growth factor (EGF) (10 ng/ml) and insulin (10 mU/ml). Maximal effects were obtained between 2 and 10 microM. DNA synthesis in the presence of EGF/insulin was reduced by the 'pure' anti-androgen flutamide, but stimulated by the 'pure' progestin promegestone. In the presence of CPA, EGF and insulin, the labelling index was twice as high in GGT-positive as in GGT-negative liver cells, regardless of whether mitogens were added at 48 or 72 h. The labelling index did not differ in the GGT-positive and negative hepatocytes when CPA was omitted. These findings are consistent with the idea that CPA has tumor-promoting activity. CPA significantly induced repair synthesis in the isolated hepatocytes from both untreated and carcinogen-treated rats. This increase was detected at a concentration as low as 2 microM and maximal effects were obtained at 20 microM. These results indicate that CPA is not only a tumor-promoting, but also a genotoxic chemical, i.e. that it might also have an initiating potential.


Mitogen-induced liver hyperplasia does not substitute for compensatory regeneration during promotion of chemical hepatocarcinogenesis. Experiments were designed to determine the efficacy of different types of liver cell proliferative stimuli given during exposure to several liver tumor-promoting regimens, on the formation of foci of enzyme-altered hepatocytes. Male Wistar rats were initiated with diethylnitrosamine (150 mg/kg body wt). After a 2 week recovery period animals were subjected to promoting regimens, the resistant hepatocyte model, the phenobarbital model and the orotic acid model. While the rats were on these regimens they were given liver cell proliferative stimulus, either a compensatory type (two-thirds partial hepatectomy or a necrogenic dose of carbon tetrachloride) or a direct hyperplastic stimulus such as that induced by the primary mitogen, lead nitrate. Initiated cells so promoted by these regimens were monitored as foci of enzyme-altered hepatocytes positive for gamma-glutamyltransferase and placental glutathione S-transferase or deficient for adenosine triphosphatase. While carbon tetrachloride and partial hepatectomy-induced compensatory regeneration stimulated the promoting ability of the regimens used, direct hyperplasia could not stimulate the formation of foci and/or nodules from initiated hepatocytes. Evaluation of thymidine incorporation indicated that there was no significant difference in the extent of DNA synthesis in both the proliferative stimuli irrespective of the promoting procedure used.


Threshold dose dependence in phenobarbital promotion of rat hepatocarcinogenesis initiated by diethylnitrosamine. The dose-response of phenobarbital (PB) promotion of hepatocarcinogenesis in rats was investigated. Male F344 rats were given 1, 4, 16, 75, 300 or 1200 p.p.m. PB solutions given ad libitum as their drinking water for 39 weeks following initiation with a single i.p. injection of diethylnitrosamine (DEN) (100 mg/kg). At week 40, the incidence of hepatic tumors was increased clearly in the DEN + PB groups given 300 p.p.m. PB or above, as compared to that in the group given DEN only. Linear dose-response curves for numbers and sizes of enzyme-altered hepatic foci (gamma-glutamyl-transpeptidase or placental glutathione S-transferase positive foci) were obtained in the dose range 16-1200 p.p.m. PB. The minimum promoting dose level of PB for enzyme-altered foci, estimated from dose-response curves by the Logit model, was calculated to be 15-23 p.p.m. Thus while dose dependence was demonstrated over a large range, a threshold was evident at low doses.


Heart rate and blood pressure changes during radiofrequency irradiation and environmental heating. 1. Whole-body exposure of animals to radiofrequency radiation (RFR) can cause an increase in body temperature. 2. Responses to heating, whether due to RFR or to more conventional means, include changes in heart rate and blood pressure. 3. Although cardiovascular responses to various types of heating are similar, differences in the magnitude of changes may result from different thermal gradients within the body. 4. This review compares the effects of RFR and conventional environmental heating on heart rate and blood pressure.


Embryonic heart rate of the domestic fowl (Gallus domesticus) in a quasiequilibrium state of altered ambient temperatures. 1. The heart rates (fH) of 12-day-old embryos (young), 16- and 18-day-old embryos (late) and of 20-day-old embryos (externally pipped eggs, EP) were measured noninvasively at a temperature of 38 degrees C and after a 5 hr exposure to an ambient temperature (Ta) within the range 34-46 degrees C. 2. All embryos survived the 5 hr exposure to Ta of 42 degrees C. The lethal Ta ranged from 44 degrees C (EP eggs) to 46 degrees C (young embryos). 3. The temperature coefficient (Q10) of fH became smaller than 2 in EP eggs as Ta was decreased or increased from 38 degrees C, implying an incipient homeothermic response of fH.


Comparative analysis of cell replacement in hibernators. 1. Cell renewal in hibernators undergoes seasonal rhythm independent of the hibernation state. 2. We propose that seasonal depression of cell renewal in tissues of hibernators is caused by seasonal involution of thymus in these animals. 3. The latter is known to be involved in the control of cell proliferation. 4. The state of hibernation per se has also an effect on cellular proliferation. 5. It induces the block of cells in the permitotic phase. It is suggested that the blockage of cells in renewing tissues of hibernators under natural deep hypothermia throughout a period of torpidity represents the adaptive reaction of the organism.


Comparative physiological responses to stressors in animals. 1. The species-specific experimental response to stressors (SSERTS) analysis has been applied to a number of species under varied short and long term conditions. 2. The measure provides quantitative data relating to the physiological responses of animals when exposed to stressors and results are presented comparing these for different methods of immobilization, euthanasia, etc. at intra- and inter-species level. 3. It is suggested that the SSERTS measure is of greater value for measuring the responses of animals to stressors than is the measurement of the concentration of single blood variables.


Decapitation or starvation raise haemolymph ecdysteroid titres in the ovoviviparous female cockroach, Blaberus craniifer Burm. 1. Decapitating newly emerged Blaberus craniifer females near the prothorax severs connections between the suboesophageal and prothoracic ganglia, thus depriving them of the neuroendocrine cephalic complex (including brain and suboesophageal ganglion) and the anterior end of prothoracic glands (PGs). 2. As demonstrated by enzyme immunoassay (EIA), headless females have higher levels of ecdysteroids (ECDs) in haemolymph than starved or fed females, indicating that the neuroendocrine cephalic complex influences circulating ECD levels. 3. The time course of hormonal peaks in decapitated females resembles that in starved females during the first post-ecdysial week, suggesting that some as yet unknown regulating mechanism of ECD production lies outside the head. 4. It is suggested that: (a) The PGs are sites for ECDs production in the early post-imaginal period, (b) the prothoracic and suboesophageal ganglia (linked by nerves to PGs) regulate PGs activity, possibly via neural inputs.


The effects of chronic exposure to elevated environmental temperature on intestinal morphology and nutrient absorption in the domestic fowl (Gallus domesticus). 1. Exposure of growing broiler chickens to elevated environmental temperature (35 degrees C) for two weeks, markedly reduced food intake (29%) and growth rate (37%) compared to birds maintained at 22 degrees C. 2. These changes in growth were accompanied by increased in vivo jejunal uptakes of galactose (36%) and methionine (50%) measured per unit intestinal dry weight. 3. Both the electrogenic (phloridzin sensitive) and non-electrogenic (phloridzin insensitive) components of galactose absorption were increased by 24 and 52% respectively during the chronic heat stress. 4. The size of the absorptive compartment may be reduced by the heat stress as reflected by decreased villus heights (19%) and wet (26%) and dry (31%) weights per unit length of jejunum. 5. It is suggested that the changes in hexose and amino acid during chronic exposure to elevated ambient temperature may reflect adaptations to optimise nutrient absorption in the face of reduced nutrition and decreases in the size of the absorptive compartment. A functional hypothyroidism (plasma luminal T3 decreased by 66%) associated with heat stress may contribute to the observed alterations in jejunal structure and function.


Amino acids and serotonin in Limax maximum after a tryptophan devoid diet. 1. Animals avoid diets lacking an essential amino acid, such as tryptophan (TRP), the precursor for serotonin (5-HT). 5-HT is important in the control of feeding. 2. To study the effects of TRP deprivation, slugs were fed TRP-devoid (DEV) or control (COR) diets. 3. Food intake was depressed in DEV, as expected, but after 2 weeks, the serontonergic metacerebral giant cell in DEV was still functional. 4. Neither brain 5-HT nor plasma TRP concentration was affected. 5. Compared with food-restricted animals that had reductions in most amino acids, the DEV group sustained a marked plasma amino acid imbalance.


Relationships between feeding, digestion and water balance in a carnivorous vertebrate, the toad Bufo bufo L. 1. Toads fed meal worms retained water, amounting to about the mass of the meal. 2. Secretion of gastric juice in the initial phase of digestion of a large meal would deplete the extracellular fluid of about half of the chloride ions. 3. Ingestion and rapid digestion of large meals in toads and other carnivorous vertebrates imply tolerance of large variations in anion composition and acidity of the extracellular fluid.


Effect of early postnatal long-term fasting on the development of peptide hydrolysis in chicks. 1. Early development of peptide hydrolysis in the digestive tract was investigated in experiments with fasted and fed ad lib. chicks during the first decade of postnatal period. 2. Pancreatic carboxypeptidase A (CPA) activity was maximal at the moment of hatch. On the second day CPA activity considerably diminished in starved and fed animal groups; further starvation (3-4 days) led to the significant increase of CPA total and specific activity, whereas the amount of enzyme in pancreas of fed chicks was rather low. 3. Aminopeptidase (AP) activity of the small intestinal surface was less sensitive to starvation. The increase of activity in all intestinal parts was observed only on the 4th day of fasting. The most sensitive to starvation were dipeptidases. Changes in their activity (2-fold increase) were detected after 24 hr of starvation. 4. The formation of specific physiological proximo-distal gradient of intestinal exopeptidase activities began only after the moment of the first feeding. 5. This gives evidence that the development of peptide hydrolysis depends not only on the age of the animal but also on the normal physiological beginning of the process of exogenous nutrition.


Effect of gamma-linolenic acid on lipid metabolism in laying hens. 1. Single comb white leghorn laying hens were given diets with additional mould, Mucus circineloides, containing gamma-linolenic acid (GLA) at levels of 0, 2.59 and 5.06 g GLA/kg diet ad lib. for 2 weeks and serum lipid contents were determined in experiment 1. 2. Serum low density lipoprotein and chylomicron levels were significantly reduced with the increase of dietary GLA levels. Serum triglyceride and cholesterol tended to be lowered by dietary GLA, but not significantly different. 3. Effects of mould GLA and extracted oil GLA on the egg yolk cholesterol concentration and fatty acid composition were compared in experiment 2. Both mould GLA and extracted oil GLA diets containing 5.32 and 4.71 g GLA/kg diet were given ad lib. for 2 weeks. 4. Yolk cholesterol content was not affected by dietary GLA sources. Content of GLA in the yolk was not altered, although that of arachidonic acid was enhanced by dietary GLA supplementation, particularly by the extracted oil GLA. 5. It is suggested that GLA is rapidly metabolized to arachidonic acid in the body and incorporated into the yolk.


Lipid and tocopherol composition of farm-raised striped and hybrid striped bass. 1. Hybrid striped bass (HB) were heavier and fatter than striped bass (SB) at harvest. 2. Total lipid, triacylglycerol and phospholipid fractions from muscle tissue of HB were characterized as having significantly larger quantities of polyunsaturated fatty acids than SB, while SB contained larger quantities of omega 3 fatty acids (specifically 22:6 and 20:5) than HB. 3. HB muscle tissue contained 14.99 and 11.42 micrograms/g dry weight of alpha- and gamma-tocopherol, respectively, while SB muscle tissue contained 38.77 and 7.15 micrograms/g dry weight of alpha- and gamma-tocopherol, respectively.


Gastrin metabolism in neonatal pigs and grower-pigs. 1. Half-life (1.7 +/- 0.1 min), distribution volume (146 +/- 12 ml/kg) and metabolic clearance rate (28 +/- 1 ml/kg/min) of little gastrin (G17) in neonatal pigs (N = 6; 3-12 days old) were significantly different from those in grower-pigs (N = 4; 161-170 days old) (2.4 +/- 0.1 min; 58 +/- 2 ml/kg; 7.9 +/- 0.3 ml/kg/min, respectively). 2. Half-life (33 +/- 4 min) and distribution volume (265 +/- 33 ml/kg) of big gastrin (G34) in neonatal pigs were greater but not significantly different from those in grower-pigs (24 +/- 2 min; 217 +/- 20 ml/kg, respectively). 3. Half-life of G17 in liver extracts from pigs 2-90 days old (40.4 +/- 4.2 min) was significantly longer than in kidney extracts (22.0 +/- 1.7 min). Half-lives of G34 in liver and kidney extracts from pigs 10-90 days old (78 +/- 6; 74 +/- 4 min, respectively) were significantly shorter than the corresponding values for 2-day-old pigs (134 +/- 3; 149 +/- 9 min, respectively). 4. Since G34 is the major circulating form of gastrin in neonatal pigs the relative longer half-life of G34 to G17 in these animals may contribute to the higher circulating gastrin concentration compared with that in older animals.


Oral absorption of biologically active insulin in common carp (Cyprinus carpio L.). 1. Bovine insulin dissolved in 0.05 M deoxycholic acid was absorbed through oral intubation in fish weighing 200-300 g. 2. The peak of absorption appeared in all fish, 30-45 min after intubation and was followed by a gradual decrease. The extent of absorption was extremely variable, with up to a 20-fold difference between individuals. No detectable insulin was found in fish intubated with vehicle. 3. The absorbed hormone retained 17-88% of its lipogenic bioactivity in vitro. The absorbed insulin lowered the levels of several amino acids in the intubated fish, indirectly indicating that its bioactivity was also retained in vivo.


An in vitro gas equilibration method for determination of chemical partition coefficients in fish. 1. A gas equilibration method for the determination of in vitro chemical partition coefficients in mammals was adapted for use with fish. 2. In vitro blood: water and tissue: blood partition coefficients were determined for three chlorinated ethanes in rainbow trout (Oncorhynchus mykiss). 3. In vitro partition coefficients accurately predicted chemical concentrations in tissues of exposed trout.


Uptake of uric acid and p-aminohippurate (PAH) by renal cortical slices of various mammals. 1. Accumulation of uric acid and PAH was measured in renal cortical slices of various mammalian species. 2. The slice to medium ratio of uric acid was above unity in the rabbit, guinea pig, pig and cow, suggesting an active accumulation of uric acid, while it was near or below unity in the rat and mongrel dog. 3. Uric acid uptake in the rabbit, guinea pig and cow was significantly inhibited by PAH. 4. Uric acid was a potent inhibitor of PAH uptake in the rabbit, guinea pig, dog and pig, but much less potent in the rat and cow. 5. Kinetic analysis showed that uric acid inhibited PAH uptake in a competitive manner in all species studied except for the cow showing a noncompetitive type. 6. These results indicate that uric acid and PAH share a common transport mechanism at the basolateral membrane of the rabbit, guinea pig and pig.


The primary culture of mouse adipocyte precursor cells in defined medium. 1. A defined medium supporting the proliferation and differentiation of adipocyte precursors isolated from inguinal fat pads of 8-12-day-old mice was developed. 2. It consists of a 1:1 mixture of DME and WAJC404A media supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), fibroblast growth factor (10 ng/ml) and high density lipoproteins (HDL) (90 micrograms protein/ml). 3. DME-F12 medium (1:1 mixture) used as a nutrient mixture in the defined medium of rat and human adipocyte precursors was inadequate for cultivating mouse adipocyte precursors. 4. HDL had a definite beneficial effect on both preadipocyte growth and differentiation. 5. Differentiation was enhanced by addition of dexamethasone (10(-9) M) but could be almost completely inhibited by transforming growth factor beta 1 (TGF-beta 1). 6. TGF-beta 1 was shown to be effective only when present in the early stages of differentiation.


Stabilization of the adenylate energy charge in erythrocytes of rats and humans at high altitude hypoxia. 1. Stabilization of adenylate energy charge and control of adenylate pool were analysed in the erythrocytes of the rat and the human exposed to highly hypoxic conditions. 2. Red cell energy charge was decreased in the rats exposed to a simulated altitude of 5000-8000 m, and then recovered to the normal value with the depletion of adenylate pool. 3. The energy charge and the adenylate pool size of the human erythrocytes did not show any change under highly hypoxic conditions. 4. Anaerobic incubation of rat erythrocytes caused a marked decrease in the energy charge, and its recovery was accompanied by the depletion of total adenylates. 5. The energy charge and total adenylates of human red cells did not change under the anaerobic incubation of erythrocytes. 6. These results suggest that the energy charge of rat erythrocytes can be controlled by depletion of the adenylate pool, but the adenylate degradation is not responsible for the stabilization of the energy charge in human erythrocytes.


Evidence for angiotensin-like molecules in the central nervous system of the leech Theromyzon tessulatum (O.F.M.). A possible diuretic effect. 1. Cells in the central nervous system of the leech Theromyzon tessulatum were revealed with an antiserum against angiotensin II. Among these cells, a group of 4-5 pairs of neurons, called beta giant cells, and located in the posterior compartments of the supraesophageal ganglion was particularly investigated. 2. The amount of angiotensin II-like substance(s) in the brain increased notably in the days immediately following the third meal. 3. Injections of angiotensin II, fragments 1-4 or 5-8 or angiotensin II and of angiotensin III into stage 3 leeches showed that fragment 5-8 of angiotensin II was the most effective: it provokes a loss of mass of the leeches, which could express a diuretic effect.


Dendritic and sustained shifts in potential to electrical stimulation of the anuran tectal surface. 1. Recordings of dendritic potentials and sustained potential shifts (SPS) were made from the brain of immobilised frogs during surface tectal electrical stimulation. 2. Single pulses evoked dendritic responses; trains caused decay of dendritic responses on the background of the evoked SPS. 3. The tectal surface SPS declined with distance from the stimulating electrode. 4. The negative surface SPS declined with tectal depth to ca 300 microns, then reversed polarity and increased in amplitude with depth up to 700 microns.


Taste synergism between monosodium glutamate and 5'-ribonucleotide in mice. 1. Strain differences of mice were found in the taste synergism between monosodium L-glutamate (MSG) and disodium 5'-guanylate (GMP). 2. Magnitudes of chorda tympani responses to the mixture of MSG and GMP over the sum of responses to each component were greater in the order of C3H/HeSlc(C3H) greater than C57BL/6CrSlc(C57BL) greater than BALB/cCrSlc(BALB) mice. The greatest synergism was observed in response to the mixture of 0.03 M MSG and 0.1 mM GMP, to which responses were about 2.6, 1.8 and 1.4 times greater than the sum of each component in C3H, C57BL and BALB mice, respectively. 3. Magnitudes of inhibition of MSG and mixture responses by the lingual treatment of proteolytic enzyme, Pronase E, were greater in the same order of C3H greater than C57BL greater than BALB mice as that observed in magnitudes of the synergism. These results suggest that there exists quantitative differences in receptors responsible for taste synergism between MSG and GMP among three mouse strains.


Hypothalamic preprosomatostatin messenger ribonucleic acid expression in mice transgenic for excess or deficient endogenous growth hormone. The influence of altered endogenous GH status on somatostatin (somatotropin release-inhibiting hormone; SRIF) gene expression was studied in two transgenic mouse models. Transgenic dwarf mice carried the rat GH gene promoter fused to the diphtheria toxin A-chain gene, placing toxin expression under GH promoter control. As a result, the toxic product of the transgene ablated all GH-expressing cells, resulting in undetectable circulating GH, reduced weight (10.6 +/- 1.0 g for transgenic dwarfs vs. 29.5 +/- 1.7 g for controls; P less than 0.001), and no detectable somatotrophs. Transgenic giant mice contained a construction combining a widely expressed metallothionein promoter and the human GH-releasing hormone (hGHRF) structural gene. Transgene expression of hGHRF resulted in overproduction of endogenous mouse GH in the anterior pituitary and weight increases (42.7 +/- 2.7 g for giants vs. 29.5 +/- 1.7 g for controls; P less than 0.005). Using in situ hybridization, control mice, transgenic dwarfs, and transgenic giants were compared for levels of prepro-SRIF mRNA. Hybridization signal intensities for prepro-SRIF mRNA were similar in transgenic dwarfs to those in littermate nontransgenic mice in non-GH-regulating regions of the brain, such as cortex (control, 31 +/- 2 U; dwarf, 27 +/- 2) and reticulothalamic nucleus (control, 41 +/- 2 U; dwarfs, 39 +/- 3). Transgenic giant mice had hybridization intensity of SRIF mRNA similar to that of normals in cortex (controls, 31 +/- 2 U; giant, 27 +/- 1) and reticulothalamic nucleus (controls, 41 +/- 2 U; giant, 40 +/- 4). In the GH-regulating neurons of the anterior periventricular hypothalamus (PeN), prepro-SRIF mRNA signal in transgenic dwarf mice decreased to 60% of that in controls (88 +/- 13 U for dwarfs vs. 147 +/- 17 U for controls; P less than 0.01), although the numbers of mRNA-expressing cells in the PeN were not different between the transgenic dwarfs and controls (dwarfs, 69 +/- 6 cells; controls, 72 +/- 4 cells). The transgenic giant mice had 230% higher prepro-SRIF mRNA signal than control mice in the PeN (343 +/- 30 U in giants vs. 147 +/- 17 U in controls; P less than 0.001). Again, the numbers of mRNA-expressing cells were not different in giants (57 +/- 9) and normals (72 +/- 4). These results suggest that while the lack of endogenous GH is accompanied by a slight decrease in transcriptional expression of SRIF in the PeN, the overproduction of endogenous GH greatly stimulates hypothalamic SRIF steady state mRNA levels.


Time-dependent reduction and potentiation of growth hormone (GH) responsiveness to GH-releasing factor induced by exogenous GH: role for somatostatin. Exogenous GH is known to exert a negative feedback effect on its own responsiveness to GH-releasing factor (GRF); however, the mechanism is not known. In the present study we examined the time course of effects of a single sc administration of recombinant human (rh) GH on GH responsiveness to GRF and investigated the possible involvement of somatostatin (SRIF) in this response. Free-moving adult male rats were administered 200 micrograms rhGH, sc, at 0800 h and subsequently challenged with 1 microgram GRF-(1-29)NH2, iv, at times of spontaneous peaks (1100 and 1500 h) and troughs (1300 h) in GH secretion during a 6-h (1000-1600 h) sampling period. H2O-injected control rats exhibited the typical cyclic responsiveness to GRF stimulation, with GRF-induced GH release significantly greater during peak compared to trough periods of the GH rhythm. Pretreatment with rhGH 3 h before GRF injection markedly inhibited the GH response to GRF at a peak time [integrated GH release over 30 min, 1135 +/- 271 vs. 6372 +/- 1185 ng/ml.30 min in H2O-injected controls (mean +/- SE); P less than 0.01]. In striking contrast, 5 h after rhGH administration, there was a 6-fold augmentation of GH responsiveness to GRF compared to that in H2O-injected controls at a trough time (7032 +/- 1622 vs. 1128 +/- 216 ng/ml.30 min; P less than 0.01). High GH responsiveness to GRF was preserved 7 h after rhGH injection. Passive immunization of rhGH-treated rats with SRIF antiserum reversed the rhGH-induced blunted GH response at 3 h (7985 +/- 366 vs. 1705 +/- 431 ng/ml.30 min in rhGH-treated control rats given normal sheep serum; P less than 0.01) and completely restored GH responsiveness to levels as high as those in H2O-injected controls. These results demonstrate that 1) a single sc injection of rhGH markedly attenuates GH responsiveness to GRF acutely for about 3 h, but subsequently enhances somatotroph sensitivity to the stimulatory actions of GRF; and 2) the short term blunting of GRF-induced GH release by rhGH is due at least in part to increased release of endogenous SRIF. The subsequent potentiation of GH responsiveness to GRF is probably due to a SRIF-mediated build-up of pituitary GH stores in a readily releasable pool. Such a mechanism of GH autofeedback may play a physiological role in the genesis of pulsatile GH secretion.


Modulation of glucocorticoid induction of tyrosine aminotransferase gene expression by variations in cell density. Glucocorticoids induce tyrosine aminotransferase (TAT) in hepatoma cells. We have previously shown that both the concentration of the agonist dexamethasone (Dex) required for half-maximal induction (EC50) and the amount of agonist activity produced by the antagonist dexamethasone 21-mesylate (Dex-Mes), expressed as a percentage of maximum induction achieved by Dex, are different in Fu5-5 and HTC cells. Furthermore, both activities vary over several weeks in each cell line in an apparently random manner, but, nevertheless, are correlated by a linear semilog plot. We now find that this long term and previously unpredictable variation in both the Dex EC50 and the amount of Dex-Mes agonist activity for the induction of TAT enzyme activity can be made to occur reproducibly in 40 h or less by changing the cell density and/or amount of medium in the tissue culture plates. Thus, a higher cell density and/or a lower volume of medium produced both higher amounts of Dex-Mes agonist activity and lower EC50 values for Dex. Experiments with cells at different densities but exposed to the same medium indicated that cell density was the dominant determinant. A qualitatively identical modulation was seen at the level of TAT mRNA, but not mouse mammary tumor virus RNA. We are not aware of any previous report of cell growth conditions altering either the level of agonist activity of an antisteroid or the EC50 of a full agonist. These results further indicate that extrachromosomal parameters, such as cell-cell contact and/or a diffusable factor(s), can modulate the basic features of glucocorticoid induction of some, but not all, glucocorticoid-inducible genes.


Autonomic nervous system mediation of the pancreatic polypeptide response to insulin-induced hypoglycemia in conscious rats. To investigate the neural regulation of pancreatic polypeptide (PP) secretion during hypoglycemia in the rat, insulin was administered to chronically cannulated rats, and plasma PP responses were compared between saline-treated animals and animals pretreated with a ganglionic blocking agent (hexamethonium), a muscarinic antagonist (atropine), combined alpha- and beta-adrenergic receptor blockade (propranolol + tolazoline), or combined adrenergic blockade + atropine. PP was measured using a new RIA which selectively detects PP in rat plasma. In control rats (n = 10), plasma PP increased from a baseline level of 30 +/- 3 pg/ml to 271 +/- 41 pg/ml during hypoglycemia (plasma glucose = 29 +/- 2 mg/dl) (delta PP = +241 +/- 42 pg/ml, P less than 0.0005), demonstrating that in rats, as in other species, insulin-induced hypoglycemia is a potent stimulus for PP release. PP only increased by 31 +/- 10 pg/ml during similar hypoglycemia in 7 hexamethonium-treated rats (P less than 0.01 vs. control animals). Thus, at least 90% of the PP response to hypoglycemia is neurally mediated. The plasma PP response to hypoglycemia was +85 +/- 24 pg/ml in atropine-treated rats (P 0.01 vs. control rats), suggesting that approximately 65% of the PP response is mediated via muscarinic acetylcholine receptors on the islet F cell. The PP response to hypoglycemia in rats with combined adrenergic blockade (delta = +168 +/- 32 pg/ml) was slightly, but not significantly smaller than that in control rats. The combination of combined blockade + atropine resulted in a PP response (delta = +26 +/- 7 pg/ml) to hypoglycemia that was similar to that in hexamethonium-treated rats (P less than 0.01 vs. control rats). These results suggest: 1) The PP response to hypoglycemia is predominantly the result of muscarinic, cholinergic activation. 2) There is a minor adrenergic contribution to the response. 3) The plasma PP response may be useful as an index of autonomic neural input to the islet during hypoglycemia.


Regulation of intermediate pituitary corticotropin-releasing hormone receptors by dopamine. It has been shown that chronic cold exposure results in selective CRH receptor up-regulation in the intermediate pituitary. Since the intermediate pituitary is under dopaminergic control, the participation of a dopaminergic mechanism in the effect of cold stress was studied in rats treated with dopaminergic agonists and antagonists. CRH receptors were measured by the binding of radioiodinated Tyr-ovine (o) CRH to neurointermediate pituitary membranes of slide-mounted sections. Cold exposure for 60 h caused the expected increase in CRH binding in neurointermediate lobe membranes. Administration of the dopaminergic agonist bromocriptine did not prevent the effect of cold stress, but increased CRH binding in control rats. The dopaminergic antagonist metoclopramide decreased intermediate pituitary CRH binding in control and cold-exposed rats. Bromocriptine administration for 1-8 days caused a progressive increase in the binding of [125I]Tyr-oCRH in neurointermediate pituitary membranes, despite atrophy of the intermediate zone. Scatchard analysis of the binding data indicated that the changes were due to variations in receptor concentration, without changes in affinity. No changes in anterior pituitary CRH receptors were observed with agonist or antagonist treatment. Autoradiographic analysis of CRH binding after 3 days of treatment with bromocriptine or haloperidol confirmed the results observed in membranes and demonstrated that changes in binding were confined to the intermediate lobe. The functional consequences of the changes in CRH binding were studied by analysis of adenylate cyclase activity in cells and homogenates of intermediate pituitaries of rats treated with bromocriptine. In 18-h cultured intermediate pituitary cells from rats treated with bromocriptine for 3 days, CRH-stimulated cAMP production, measured in the presence of phosphodiesterase inhibitors, was increased to levels only slightly higher than those in cells from control rats. Likewise, CRH-stimulated adenylate cyclase, measured by conversion of [32P]ATP to [32P] cAMP, was not significantly different in homogenates from microdissected intermediate lobes from control and bromocriptine-treated rats. The lack of parallel changes in adenylate cyclase responsiveness suggests only partial receptor coupling, probably reflecting an inhibitory effect of dopamine on components of the adenylate cyclase. This study demonstrates that in contrast to the recognized inhibitory effect on cell division and POMC mRNA expression, dopamine causes up-regulation of CRH receptors in the intermediate pituitary. The qualitatively similar and nonadditive effects of cold stress and dopaminergic agonists suggest that a dopaminergic mechanism may be involved in intermediate pituitary CRH receptor regulation during chronic cold stress.


Aging enhances inhibitory action of somatostatin in rat pancreas. This study examines the effects of donor age on exogenous somatostatin inhibition of insulin secretion stimulated by 10 mM D-glyceraldehyde and by 20 mM beta-D-glucose in isolated perfused rat pancreas. Both 6 and 30 nM synthetic somatostatin-14 affect both glyceraldehyde- and glucose-stimulated insulin secretion to a greater degree in pancreases from old animals (24-27 months) than in those from young (2-5 months). We conclude that an increased sensitivity to inhibitory actions of somatostatin during aging, observed in pituitary tissues in vitro, occurs in pancreatic tissues as well and may constitute a general phenomenon in tissues subject to somatostatin inhibition.


A transferable silencing domain is present in the thyroid hormone receptor, in the v-erbA oncogene product and in the retinoic acid receptor. Inhibition of gene transcription is brought about by several mechanisms. The least understood mechanism is probably silencing, the analogue to transcriptional enhancing. We provide evidence that the silencing function of the oncogene product v-ERBA or the cellular counterpart, the thyroid hormone receptor (TR, c-erbA) is located in the C-terminal part and is transferable to a heterologous DNA binding domain. Deletion analyses suggest an important role for a basic and hydrophilic amino acid stretch on both ends of the domain. In addition we show that the related retinoic acid receptor (RAR) also contains a functional silencing domain similar in size and amino acid sequence. However, the activity of this domain can be neutralized by an additional domain in the C-terminus which functions cell specifically.


A subdomain in the transmembrane domain is necessary for p185neu* activation. The neu proto-oncogene encodes a protein highly homologous to the epidermal growth factor receptor. The neu protein (p185) has a molecular weight of 185,000 Daltons and, like the EGF receptor, possesses tyrosine kinase activity. neu is activated in chemically induced rat neuro/glioblastomas by substitution of valine 664 with glutamic acid within the transmembrane domain. The activated neu* protein (p185*) has an elevated tyrosine kinase activity and a higher propensity to dimerize, but the mechanism of this activation is still unknown. We have used site-directed mutagenesis to explore the role of specific amino acids within the transmembrane domain in this activation. We found that the lateral position and rotational orientation of the glutamic acid in the transmembrane domain does not correlate with transformation. However, the primary structure in the vicinity of Glu664 plays a significant role in this activation. Our results suggest that the Glu664 activation involves highly specific interactions in the transmembrane domain of p185.


Antp-type homeodomains have distinct DNA binding specificities that correlate with their different regulatory functions in embryos. Much of the functional specificity of Drosophila homeotic selector proteins, in their ability to regulate specific genes and to assign specific segmental identities, appears to map within their different, but closely related homeodomains. For example, the Drosophila Dfd and human HOX4B (Hox 4.2) proteins, which have extensive structural similarity only in their respective homeodomains, both specifically activate the Dfd promoter. In contrast, a chimeric Dfd protein containing the Ubx homeodomain (Dfd/Ubx) specifically activates the Antp P1 promoter, which is normally targeted by Ubx. Using a variety of DNA binding assays, we find significant differences in DNA binding preferences between the Dfd, Dfd/Ubx and Ubx proteins when Dfd and Antp upstream regulatory sequences are used as binding substrates. No significant differences in DNA binding specificity were detected between the human HOX4B (Hox 4.2) and Drosophila Dfd proteins. All of these full-length proteins bound as monomers to high affinity DNA binding sites, and interference assays indicate that they interact with DNA in a way that is very similar to homeodomain polypeptides. These experiments indicate that the ninth amino acid of the recognition helix of the homeodomain, which is glutamine in all four of these Antp-type homeodomain proteins, is not sufficient to determine their DNA binding specificities. The good correlation between the in vitro DNA binding preferences of these four Antp-type homeodomain proteins and their ability to specifically regulate a Dfd enhancer element in the embryo, suggests that the modest binding differences that distinguish them make an important contribution to their unique regulatory specificities.


Cell surface marker analysis of mouse thymic dendritic cells. Cell surface markers of mouse thymic dendritic cells have been studied by flow cytometry after isolation by collagenase digestion, separation of the low-density cell fraction and differential adherence. The dendritic cell preparation had a purity of greater than 90%, the contaminating population being essentially composed of thymocytes, macrophages constituting less than 1%. Dendritic cells displayed high forward and low-intermediate side angle scatter, and expressed high levels of major histocompatibility complex (MHC) class I and class II molecules, the heat-stable antigen (HSA), the adhesion molecules Pgp-1 (CD44), LFA-1, ICAM-1 and low levels of Mac-1 and the leukocyte common antigen CD45. Thymic dendritic cells are negative for the stem cell antigen-2 (Sca-2), the B cell-specific form of CD45 (B220), the mouse macrophage markers Fc receptor and F4/80, and the granulocyte marker Gr-1. However, although they do not express the T cell markers Thy-1, CD2, CD3, CD4 and CD5, 20%-30% of dendritic cells are positive for the interleukin 2 receptor alpha chain (CD25), and about 30% express intermediate levels of CD8. These results are discussed with regard to the functional significance of the expression of CD8 by thymic dendritic cells, and the existence of different dendritic cell subpopulations in the murine thymus.


Overexpression of multidrug resistance-associated p170-glycoprotein in acute non-lymphocytic leukemia. Resistance to several cytotoxic agents, including anthracyclines, vinca alkaloids and epipodophylline derivatives (multidrug resistance, or MDR) can develop in tumor cells by overexpression of a 170-kd glycoprotein (p170) which is an essential component of a membrane transport system leading to increased drug efflux and decreased intracellular drug concentration. By means of a p170-directed monoclonal antibody (MRK-16) and immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique), we investigated the expression of p170 in marrow blast cells of 59 cases (38 at diagnosis and 21 in relapse) of acute-non-lymphocytic leukemia (ANLL). The proportion of strongly MDR-positive cells was higher in relapse that at diagnosis (median 15.5% vs 1.5%). Out of 31 patients who were evaluable for the results of first remission induction, failure of first-line treatment (including Daunorubicin, standard-dose and high-dose Arabinosyl Cytosine, and sometimes also Mitoxantrone) occurred in 8/22 MDR-positive cases and in 1/9 MDR-negative ones (p = 0.21). Failure of first-line treatment was always associated with a progressive increase of p170 expression. Total failures (no remission plus early relapse) were more frequent (p = 0.001) among MDR-positive cases (16/22) than among the others (2/9). These data show that MDR is very frequent in ANLL also at diagnosis and suggest that MDR can contribute to early failure of standard treatment.


The LIM domain-containing homeo box gene Xlim-1 is expressed specifically in the organizer region of Xenopus gastrula embryos. A novel cysteine-rich motif, named LIM, has been identified in the homeo box genes lin-11, Isl-1, and mec-3; the mec-3 and lin-11 genes determine cell lineages in Caenorhabditis elegans. We isolated LIM class homeo box genes from Xenopus laevis that are closely related to lin-11 and mec-3 in the LIM and homeo domains. This paper deals with one of these genes, Xlim-1. Xlim-1 mRNA is found at low abundance in the unfertilized egg, has a major expression phase at the gastrula stage, decreases, and rises again during the tadpole stage. In adult tissues the brain shows the highest abundance, by far, of Xlim-1 mRNA. The maternal and late expression phases of the Xlim-1 gene suggest that it has multiple functions at different stages of the Xenopus life cycle. In the gastrula embryo, Xlim-1 mRNA is localized in the dorsal lip and the dorsal mesoderm, that is, in the region of Spemann's organizer. Explant experiments showed that Xlim-1 mRNA is induced by the mesoderm-inducer activin A and by retinoic acid, which is not a mesoderm inducer but affects patterning during Xenopus embryogenesis; application of activin A and retinoic acid together results in synergistic induction. The structure, inducibility, and localized expression in the organizer of the Xlim-1 gene suggest that it has a role in establishing body pattern during gastrulation.


Inhibitory effects of nicotinamide on recombinant human interferon-gamma-induced intercellular adhesion molecule-1 (ICAM-1) and HLA-DR antigen expression on cultured human endothelial cells. Intercellular adhesion molecule-1 (ICAM-1), HLA-A, B, C and HLA-DR antigen on endothelial cells (EC) play important roles in the development of inflammatory processes in autoimmune disorders. In the present study, we investigated the effect of nicotinamide, an inhibitor of poly(ADP ribose) synthetase, on interferon-gamma (IFN gamma)-induced ICAM-1 and HLA-DR antigen expression on the surface of cultured human umbilical vein endothelial cells, assessed by flow cytometry, and EC proliferation by counting cell numbers and [3H]thymidine incorporation assays. Nicotinamide dose-dependently inhibited the IFN-gamma-induced ICAM-1 and HLA-DR antigen expression, but not HLA-A, B, C antigen expression on cultured EC. Furthermore, nicotinamide significantly inhibited endothelial cell proliferation, as assessed by [3H]thymidine incorporation assay. Our findings suggest that nicotinamide may suppress mononuclear cell infiltration, antigen presentation and angiogenesis in the lesions of autoimmune disorders by reducing both IFN gamma-induced ICAM-1 and HLA-DR antigen expression on EC, and EC proliferation. Therefore, nicotinamide can be used for the treatment and prevention of the development of autoimmune disorders.


Flow cytometric analysis of the expression of murine B and T surface markers from birth to adulthood. The proportion of nucleated splenocytes bearing B-lymphocyte markers B220, surface IgM (sIgM) and sIgD, as well as the T-lymphocyte markers Thy 1.2, CD5, CD8a and CD4 were quantitated by flow cytometric analysis (FACS) throughout postpartum development in the A/J mouse. Full expression of B lymphocyte markers was achieved much sooner than expression of T lymphocyte markers. This was especially true for B220, which was found on 8% of all splenocytes at day 5 and reached adult levels (47-50%) by weaning at day 22. Expression of sIgM and sIgD were 13% and 9%, respectively, of all splenocytes at day 5 with mature levels not expressed until day 35 postpartum (approximately 36% of cells were positive for these markers). T lymphocyte markers, on the other hand, did not reach full expression until sexual maturity. For example, Thy 1.2 expression was 8% on day 5 and did not reach mature levels (28-30%) until day 56. CD5 closely paralleled Thy 1.2 expression rising from only 2% on day 5 to 27% by day 56. Likewise, CD8a and CD4 marker development paralleled one another with CD8a rising from 1% on day 5 to 10% by day 56 and CD4 rising from 5% on day 5 to 19% by day 56. These data demonstrate the variability in the time of appearance and rate of maturation of the various lymphocyte cell surface markers during postpartum development. They also serve as a reference to identify alterations in lymphocyte development created by immunodeficiency diseases.


Application of anti-CD3-treated lymphocytes as stimulator cells in human autologous mixed lymphocyte cultures for generation of autorestricted CD8+ suppressor T cells. The purpose of the present study was to investigate the application of anti-CD3-treated lymphocytes as stimulator cells in human one-way autologous mixed lymphocyte cultures (MLC) for the generation of suppressor cells. MLC-activated CD4-CD8+ CD16- T cells were non-cytotoxic, while they down-regulated the proliferation of autologous (but not allogeneic) responder lymphocytes in allogeneic test MLC.


T cell receptor gamma/delta expression on lymphocyte populations of breast cancer patients. The quantitative distribution and phenotype of gamma/delta lymphocytes in the peripheral blood (PBL), tumour draining lymph node (LNL) and tumour infiltrating lymphocytes (TIL) from breast carcinoma patients were determined by one- and two-colour flow cytometry. The TCR-gamma/delta + cells generally expressed the T cell lineage antigen CD3. The proportions of such cells were variable but generally small from all the three sources. Phenotypic analysis revealed that the CD8 marker was consistently and predominantly observed on gamma/delta + CD3+ cells in the tumour infiltrate, whereas CD4 expression, while generally low, was noted on a significant percentage (median 10%) of LNL gamma/delta + lymphocytes. In both PBL and LNL the predominant gamma/delta cell population was CD4-8-.


The bactericidal capacity of peripheral blood monocytes from HIV positive patients may collapse very soon after the infection. The activity of peripheral blood monocytes from HIV positive patients was measured by the intensity of the chemoluminescence those cells emit when they ingest a foreign particle. In most patients (84%) that value was impaired. Such an alteration may occur very early in the course of the disease. The anti-p24 antibody titer was correlated with monocyte phagocytic potential as measured by the chemoluminescent value thus indicating the need for adequate monocyte activity in order to obtain antibody formation. The severity of opportunistic infections that HIV positive subjects may develop is a clear indication that their immune systems are abnormal. The most frequently affected cells are those which bear the viral receptor, the CD4 antigen. Those cells are mainly the helper T cells and monocytes. The monocytes are the immune system phagocytic cells which actively control infections, in part by the release oxidative radicals. Those radicals can easily be measured by the chemoluminescence (CL) the cells emit when they ingest a foreign particle. This study examines the CL emitted by peripheral blood monocytes from HIV-positive and control subjects while they phagocytose opsonized zymosan in vitro and correlates these values with other laboratory parameters.


Transitory expression of Thy-1 antigen in immature B cell lines. In cmu- B220+ Thy-1- murine immature B cells transformed with a temperature-sensitive mutant of Abelson murine leukemia virus, a low level of Thy-1 antigen was transitorily expressed after the shift of the culture temperature from the permissive (35 degrees C) to the non-permissive (39 degrees C) temperature. On the other hand, B220 antigen was persistently expressed regardless of whether the cells were cultured at the permissive or non-permissive temperature. No other T-lineage-specific antigens, CD3, CD4, and CD8 were induced during the culture at the non-permissive temperature. Expression of Thy-1 antigen was confirmed by the detection of a low level of Thy-1-specific mRNA. These results clearly showed that immature B cells could express Thy-1 antigen during proliferation and/or differentiation. The results imply a role for Thy-1 antigen in B cell proliferation and/or differentiation.


Lovastatin and coadministered antihypertensive/cardiovascular agents. Hypertension and hypercholesterolemia frequently coexist and may require concomitant drug treatments. The efficacy and safety profile of lovastatin given in the presence of antihypertensive medication was evaluated using patient subgroups identified in the Expanded Clinical Evaluation of Lovastatin (EXCEL) Study. The EXCEL study examined 8,245 patients with moderate hypercholesterolemia randomly assigned either to a group treated with lovastatin (20-80 mg daily) or to a group given placebo for 48 weeks. After adjustment for patient characteristics, pairwise comparisons were made between patients taking no antihypertensive agents (n = 3,772) and those taking either calcium antagonists (n = 446), selective beta 1-adrenergic receptor blockers (n = 326), nonselective beta-adrenergic receptor blockers (n = 219), potassium-sparing diuretics (n = 187), thiazide diuretics (n = 126), or angiotensin converting enzyme inhibitors (n = 171). The placebo-corrected dose-dependent effect of lovastatin on the percent change from baseline in low-density lipoprotein cholesterol was not attenuated in any subgroup and was slightly enhanced in the calcium antagonist subgroup (-29% to -44%, p = 0.06) when compared with patients taking no antihypertensive agents (-24% to -40%); this difference, however, was only of borderline significance. Patterns of lovastatin-induced increase in high-density lipoprotein cholesterol and decrease in triglycerides were not consistently different among the subgroups. Examination of mean changes in serum transaminases, mean changes in creatine kinase, and the proportion of patients discontinuing therapy for clinical adverse experiences did not indicate the presence of an interaction.(ABSTRACT TRUNCATED AT 250 WORDS)