Desmoplastic melanoma: patterns of recurrence.

Desmoplastic melanoma is a rare type of malignant melanoma, recognized since 1971. Other variants of desmoplastic melanoma include neural transforming melanoma and neurotropic melanoma. The pathology and clinical features of 58 patients whose tumor had the features of desmoplastic melanoma, neural transforming melanoma, and neurotropic melanoma, either separately or in combination, were examined to assess patterns of recurrent disease. The tumor was situated on the head and neck in 41% of patients and was amelanotic in 71% of patients. There was an associated superficial melanoma in 48% of patients. There was a combination of the 3 histologic patterns, commonly found in the 1 melanoma. Local recurrence occurred in 29% of patients and malignant cranial neuropathies were documented in 4 patients. Nineteen percent of patients have died from disseminated disease. Neurotropic melanomas had a lower incidence of visceral recurrence. Desmoplastic and neural transforming melanomas had similar rates of local and visceral recurrence. When this specific variant of melanoma is compared with larger series of malignant melanoma in general, they appear to be more advanced locally, with a higher incidence of local recurrence. When considered in relation to the thicker nondesmoplastic melanomas, the survival is no worse and may be more favorable. Surgeons should excise the primary tumor and local recurrences with wide margins and adopt close follow-up. On the head and neck, symptoms and signs relating to trigeminal or facial nerve innervation may herald a developing malignant cranial neuropathy.


Multiple administration with interleukin-2 potentiates antigen-specific responses to subunit vaccination with bovine herpesvirus-1 glycoprotein IV.

Interleukin-2 has been described as an effective adjuvant for a number of antigens in different host species. Previously, we demonstrated the adjuvant activity of recombinant bovine IL-2 with a glycoprotein IV (gIV) subunit vaccine from bovine herpesvirus type-1 (BHV-1). In the present study, primary antibody responses were assessed in cattle immunized with either 2 or 50 micrograms of gIV, and treated with multiple doses of IL-2 or combinations of IL-2 and IFN-alpha or IL-2 and IFN-gamma. IL-2 was able to augment significantly antibody responses detected by either ELISA or virus neutralization. More significantly, IL-2 was able to enhance antibody titres in animals immunized with only 2 micrograms gIV to levels similar to those immunized with 50 micrograms gIV in the absence of IL-2. For optimal stimulation, multiple injections of IL-2 and Avridine had to be used in the formulation; other oil adjuvants or IL-2 alone could not induce a primary serum antibody response. Addition of IFN-alpha or IFN-gamma to the IL-2/gIV/Avridine formulation did not affect any of the immune parameters tested. As IFN-alpha is an effective immunoprophylactic agent for infectious bovine rhinotracheitis (IBR), combination vaccine-immunoprophylaxis may become feasible using IL-2 as a co-adjuvant. Thus, extremely low doses of antigen and only one immunization may be an effective vaccine given in combination with interferon prophylactic treatment.


Expression of foreign epitopes on recombinant occlusion bodies of baculoviruses.

Recombinant polyhedrin proteins of the baculovirus Autographa californica nuclear polyhedrosis virus were constructed to serve as immunologic carriers of foreign epitopes. Several recombinants containing an influenza haemagglutinin epitope were obtained. Three of the five recombinants formed occlusion bodies (OBs) and two did not. All of the recombinant polyhedrin proteins reacted with a monoclonal antibody (mAb) specific for an influenza epitope in Western blots. Presentation of the foreign epitope on the surface of recombinant OBs was demonstrated by specific immunoprecipitation of the anti-influenza mAb with recombinant OBs. The recombinant polyhedrin-influenza epitope fusion protein stimulated an influenza-specific immune response in rabbits.


Palliative care and melanoma: the care of the patient with progressive disease.

The World Health Organization has taken the lead in recent years in encouraging an appropriate focus on palliative care which includes but goes beyond terminal care. Comprehensive care of patients with advanced progressive disease requires increased emphasis, with adequate resources. Management of major symptoms is a crucial component of such a care program and requires the application of recent advances in palliative therapeutics (notably but not only good quality pain management), whether or not direct antitumor strategies are being continued. The choice of treatment approaches must be related to the patient's personal priorities.


Histochemical demonstration of gastrointestinal mucins in ovarian mucinous cystadenoma.

Forty-one specimens of mucinous cystadenoma (MCA) of the ovary and 13 specimens of normal uterine endocervix were investigated histochemically using alcian blue pH 2.5-PAS, high iron diamine-alcian blue pH 2.5, galactose oxidase-Schiff reaction (GOS, for detection of gastric surface mucous cell mucins), paradoxical Concanavalin A staining (PCS, for detection of gastric gland cell mucins) and periodic acid-sodium borohydride-potassium hydroxide-PAS (PA-SB-PH-PAS) reaction (for detection of mucins of the large intestine). In addition, Grimelius staining was carried out to explore the distribution of endocrine cells. The MCAs contained abundant neutral mucins but a little acid mucins, whereas sulfomucins predominated in normal endocervices. Among the MCAs, 30, 26, 2 and 9 had tumor cells positive for GOS, PCS, PA-SB-PH-PAS and Grimelius stain, respectively. These results indicate that MCAs frequently contain tumor cells of the gastrointestinal type and endocrine cell type. Endocervical epithelia, on the other hand, lack reactivity for PCS, PA-SB-PH-PAS and Grimelius stain, although in 8 of the present specimens, the lining cells showed weak GOS reactivity.


Immunoreactive copper-zinc superoxide dismutase in damaged human myocardium.

The myocardium in 50 autopsy cases was studied using immunostaining for copper-zinc superoxide dismutase (CuZn-SOD) and standard histochemical procedures. Mucinous degeneration observed in 42 cases showed moderately enhanced expression of immunoreactive CuZn-SOD in lesions which were stained strongly by periodic acid-Schiff but negative with Heidenhain iron-hematoxylin (HIH), von Kossa and luxol fast blue (LFB) stains, whereas coagulation necrosis in 4 cases revealed almost identical immunostaining for CuZn-SOD and HIH to that of contraction band necrosis, i.e. strongly positive HIH staining but negative immunostaining. Basophilic alteration of the myocardial cells in sections fixed with 4% formalin in 2% calcium acetate was seen in 29 cases, being identified frequently in isolated cells as well as in several foci varying considerably in size. This type of alteration demonstrated significantly enhanced expression of immunoreactive CuZn-SOD and was strongly positive with von Kossa and LFB stains. The present study indicates that the myocardium can be affected by free radicals produced in any organ of the body, and that subsequently, insoluble phospholipids react with calcium ions in the fixative and accumulate in the basophilic sarcoplasm.


Sensitivity and accuracy of a bio-assay for the determination of the concentration of residual pesticide in natural water bodies.

Determination of pesticide levels in natural water bodies requires complex chemical methods not always available in tropical countries where such control programmes are implemented. Apart from chemical methods, effectiveness of treatment can be estimated by mortality of target and non-target species. But the latter criteria do not permit a good appraisal of dispersion and residual activity of pesticide. We propose here a simple and cheap bio-assay adaptable to water-borne vector or intermediate host control programmes. Results obtained with the bio-assay were compared with those obtained with gas liquid chromatography, a standard chemical method.


Effects of left ventricular dysfunction on the circadian variation of ventricular premature complexes in healed myocardial infarction.

Circadian variation in the onset of cardiovascular events including sudden cardiac death, myocardial infarction and ventricular arrhythmias has been described. The effect of left ventricular (LV) dysfunction on the circadian variation of ventricular premature complex (VPC) frequency was evaluated in 132 patients with frequent VPCs and reduced LV function after myocardial infarction. Patients were prospectively divided in 2 groups based on LV ejection fraction (EF) (those with LVEF less than or equal to 0.30, and those with LVEF between 0.30 and 0.45). Median hourly VPC frequencies and heart rates were compared between the 2 groups. Subgroup analyses based on treatment with beta-adrenoceptor blocking agents and on New York Heart Association functional class were also performed. In patients with LVEF greater than 0.30, a distinct circadian variation of VPCs, and the expected morning increase in VPC frequency were present. In contrast, a distinct circadian variation of VPCs was absent in patients with LVEF less than or equal to 0.30. A circadian variation of VPC frequency was also absent in patients with severe symptomatic congestive heart failure (New York Heart Association class III-IV). Treatment with beta-adrenoceptor blocking agents was associated with a loss of the circadian variation of VPC frequency. The circadian variation of heart rate was also blunted in the group treated with beta-adrenoceptor blocking agents. The proportion of subjects manifesting a positive correlation between heart rate and VPC frequency was lower in subjects with LVEF less than or equal to 0.30 (26%) than in those with LVEF greater than 0.30 (46%) (p less than 0.05). Thus, circadian variation of VPC frequency is absent in patients with severe LV dysfunction.


Molecular biology of African trypanosomes: development of new strategies to combat an old disease.

African trypanosomes are protozoan parasites that cause a number of diseases of man and domesticated animals in large regions of sub-Saharan Africa. The diseases have proven to be particularly difficult to prevent or to effectively treat due to features of both the trypanosome and the insect vector, the tsetse fly. The habitat of the tsetse and its resistance to insecticides have rendered vector control efforts ineffective. Attempts to develop a vaccine against the African trypanosomes has been dwarfed by the parasite's ability to change the composition of its exposed surface antigens. This process of antigenic variation allows the parasite to avoid the host's immune response and presents the host with a seemingly endless antigenic repertoire. Since conventional approaches to the control of African trypanosomiasis have largely met with failure, there has been a renewed interest in identifying novel aspects of the biology, biochemistry, and molecular biology of trypanosomes that might be exploited to develop new targets for vaccines or chemotherapy. Importantly, this research has opened a virtual Pandora's box of exciting biochemical and molecular surprises, which makes the African trypanosomes not only important medical pathogens but also an exciting experimental system for the basic scientist. In this review, the authors will describe some of the most recent and intriguing developments in the field of molecular parasitology.


The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. Extraction of protein and application to immunoblotting.

The authors have previously reported a new fixation and paraffin-embedding method (the AMeX method), which preserves many antigens as well as high molecular-weight DNA and RNA that are normally destroyed by the routine formalin fixation and paraffin-embedding process. In the present study, the authors analyzed the preservation of protein suitable for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting in tissue fixed by the AMeX method. The method used for extraction of protein from AMeX-processed tissue sections after deparaffinization was the same as that for extraction from fresh tissues. The total amount of protein extracted from 50-mg (wet weight) AMeX-processed mouse liver tissue was the same as that from fresh tissue. The electrophoretic mobility and staining intensity of protein on SDS-polyacrylamide gel, and the immunoblotting pattern and staining intensity with several antibodies, were identical for both AMeX-processed and fresh tissue. Degradation of protein was minimal for storage periods of 2 years in paraffin block. The authors also showed that pellets of cultured cells can be processed by this method for immunologic analysis. This new fixation and paraffin-embedding method is a useful tool for obtaining information on correlations between morphologic features and immunochemical and molecular biological data.


Expression of endothelial adhesion molecules in vivo. Increased endothelial ICAM-2 expression in lymphoid malignancies.

The authors have compared the reactivity of monoclonal antibodies (MAb) directed against endothelial adhesion molecules (ICAM-1, ICAM-2, VCAM-1, ELAM-1) in hyperplastic, nonmalignant, and malignant lymph nodes. The authors demonstrate that the reactivity with ICAM-1 MAb is stronger in the high endothelial venules (HEV) and other smaller vessels (SV) in lymphomas compared with hyperplastic lymph nodes. Similarly, the reactivity of an ICAM-2 MAb (6D5) was shown to be higher in malignant lymph nodes compared with nonmalignant ones. ICAM-2 MAb stained both the HEV and SV. VCAM-1 MAb reacted strongly with germinal centers and its endothelial reactivity was higher in the lymphoma nodes. ELAM-1 MAb stained only faintly some endothelial cells in malignant tissue. These data suggest that besides the known regulatable endothelial adhesion molecules ICAM-1 and VCAM-1, the expression of ICAM-2 can be modified.


Accumulation of amyloid precursor protein in neurons after intraventricular injection of colchicine.

To study a possible relationship between inhibition of axonal flow and amyloidogenesis, the authors examined amyloid precursor protein (APP) immunoreactivity in rat brain treated with colchicine. After intraventricular injection of colchicine, the proximal axons of exposed neurons became swollen and showed a large increase in APP immunoreactivity, whereas the cytoplasm and dendritic processes showed lesser increases. These changes were seen in ipsilateral neurons of the hippocampus, lateral septal nucleus, amygdala, and entorhinal, parietal and temporal cortices, as well as bilaterally in the periventricular hypothalamic nucleus. The increase of APP immunoreactivity appeared as early as 3 hours after the injection. It peaked at around 24 hours, and began to clear after about 4 days. A few strongly APP-positive dystrophic neurons remained. In serial sections at these later time periods, some strongly argentophilic neurons and Alz-50 positive neurons, each with abnormal neurities, could be demonstrated. The result suggests that APP may undergo fast axoplasmic flow in rat brain and that argentophilic changes of Alz-50 immunoproduction may follow APP accumulation caused by inhibition of axoplasmic flow.


Activation of the contact system in lethal hypotensive bacteremia in a baboon model.

The hypotension in septicemia is believed to be mediated by the combined action of many mediators including cytokines, prostaglandins, and complement components. To evaluate the contribution of the contact/kinin-forming system to hypotension, the authors used an established experimental baboon model of bacteremia in which two concentrations of Escherichia Coli (E. coli) were used to produce lethal and nonlethal hypotension. The lethal group (n = 5) developed irreversible hypotension that significantly correlated with the decline in levels of high molecular weight kininogen (HK) and an increase in alpha 2 macroglobulin-kallikrein complexes (alpha 2M-kal). The nonlethal group (n = 9) experienced reversible hypotension, a less striking decline in HK, and only slight elevation in alpha 2M-kal. No significant changes were found in levels of factor XII, prekallikrein, and factor XI in either group. A significant change in the contact system, which reflects the fatal outcome, is the rise in alpha 2M-kal. This study suggests that irreversible hypotension correlates with prolonged activation of the contact system.


Multiple ionic mechanisms of early afterdepolarizations in isolated ventricular myocytes from guinea-pig hearts.

Ionic mechanisms of early afterdepolarization (EAD) induced by the K(+)-free solution or veratridine were studied with guinea-pig ventricular myocytes using the patch-clamp technique of whole-cell and cell-attached patch configurations. In the K(+)-free solution, myocytes exhibited prolonged action potential duration with humps on the final repolarization phase, which eventually turned into EAD starting around -70 mV and induced triggered activity. Application of 0.5 mM Cd2+ inhibited the development of EAD and caused depolarization of maximum diastolic potentials around -30 mV, although Cd2+ did not prevent prolongation of the action potential. Application of 50-100 microM Ni2+ or 30 microM tetrodotoxin had little effects on EAD and diastolic potentials. The background current-voltage relation examined by a ramp voltage clamp showed inhibition of the inward rectifier K+ current, induction of steady inward current between -40 and -10 mV, and increase in the outward tail current upon repolarization in the K(+)-free solution. Cd2+ completely blocked the steady inward current at the plateau level and partially depressed the delayed outward K+ current, while Ni2+ had no effects on the background I-V relation. Tetrodotoxin showed a mild inhibitory effect on the inward component of the background current negative to -50 mV, but left the steady inward current at the plateau level. Therefore, EAD in the K(+)-free condition is mainly formed by decreased inward rectifier K+ current, activation of the L-type Ca2+ current, and time-dependent decay of the delayed outward K+ current upon repolarization. Application of 25-100 microM veratridine caused marked prolongation of action potential with appearance of regenerative EADs. Action potential prolongation and EADs were partially abolished by Cd2+ and completely eliminated by tetrodotoxin. The single channel current recordings showed a decreased current amplitude, and prolonged and delayed openings of the Na+ channel currents by veratridine. Thus, an ensemble average current showed markedly prolonged decay time constant of 609 msec in veratridine from 3.6 msec in the control. These results indicate that veratridine-induced EAD is mainly formed by altered properties of the Na+ channel current and partly by the L-type Ca2+ current due to slowed repolarization. Thus, EAD can be induced by different ionic mechanisms depending on the basal conditions.


Massive accumulation of modified tau and severe depletion of normal tau characterize the cerebral cortex and white matter of Alzheimer's disease. Demonstration using the hydrated autoclaving method.

Using the hydrated autoclaving method, a new immunohistochemical procedure to enhance tau immunoreactivity in formalin-fixed brain tissue, the authors recently reported that tau protein is detected in neuronal cell bodies and proximal dendrites, gray matter neuropil, axons, and glial cells in normal human hippocampus and neocortex. In the this study, the authors performed a comparative study of the distribution of normal and modified forms of tau in Alzheimer's disease (AD) and control brains. In the cerebral cortex and white matter of AD brains, a massive accumulation of modified tau and/or severe depletion of normal tau were documented in all the tau compartments. In mild AD cases, gray matter neuropil, axons, and glial cells were less severely involved than neuronal perikarya. In the controls, neuronal perikarya were often involved by modified tau accumulation, but the other compartments showed normal distribution. These observations suggest that modifications of tau which lead to neurofibrillary lesions in AD may begin in neuronal perikarya and extend to the other tau compartments in advanced stages of the disease.


Adenosine receptors.

Our knowledge of A1 and A2ARs has grown dramatically since they were first defined by Burnstock. With the recent purification of the A1AR and the cDNA cloning of the A2AR, an even more rapid expansion of information regarding their structure, function, and regulation should now ensue. We may then be able to return to the intact cell and organ system and better understand the physiological role of these ubiquitous receptors.


Porcine retrovirus: optimal conditions for its biochemical detection.

The assay of reverse transcriptase (RT) activity was used to detect the presence of retrovirus in porcine cells. A set of optimal assay conditions was determined to design a sensitive, quantitative and reproducible RT assay for porcine systems. The template-primer poly(rA).oligo(dT) was an absolute requirement. The presence of Mn++ was indispensable, with an optimal concentration of 0.25 mM. Monocations (K+, Na+) at 50 mM greatly enhanced, but their high doses inhibited the reaction. The pH of the medium influenced very much the reaction, especially with non-purified virus samples, with which the RT activity was inhibited at pHs above 8.2. Non-ionic detergents at 1% enhanced several-fold the RT activity. It was also shown that porcine retrovirus could be spontaneously reactivated in porcine cell lines by in vitro long-term propagation and transmitted to pigs by inoculation with virus-producing cells.


Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates.

A 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.


HIV-1 reverse transcriptase inhibition by a dipyridodiazepinone derivative: BI-RG-587.

The dipyridodiazepinone derivative 6,11-dihydro-11-cyclopropyl-4-methyldipyrido[2,3-b:2',3'-e]-[1,4] diazepin-6-one (BI-RG-587) selectively inhibits human immunodeficiency virus type 1 (HIV-1) replication by suppressing HIV-1 reverse transcriptase activity. Both RNA- and DNA-dependent polymerase associated activities of this enzyme were found to be inhibited by BI-RG-587 in a pattern dependent on the template used. The lowest IC50 values were obtained using poly(rC)-oligo(dG)12-18 and poly(dA)-oligo(dT)12-18 as template-primer. For the RNA-dependent activity poly(rC)-oligo(dG)12-18 and dGTP appeared to enhance the inhibition of the RNA-dependent enzyme activity by BI-RG-587, with the effect of poly(rC)-oligo(dG)12-18 dominating that of dGTP. Poly(rA)-oligo(dT)10 seemed to decrease the inhibition whereas poly(rU)-oligo(dA)12-18 or poly(rG)-oligo-(dC)12-18 had no effect. dATP, dTTP and dCTP, three nucleotide triphosphates, also had no impact on the inhibition. Differences were observed for the template-dependent action of BI-RG-587 against the DNA-dependent enzyme activity. Both substrates were required to allow the inhibition by BI-RG-587 in the poly(dC)-oligo(dG)12-18 and dGTP reaction, whereas only the template and enzyme interaction seemed to be necessary for the poly(dA)-oligo(dT)12-18 and dTTP reaction. The different behaviors of DNA- and RNA-dependent DNA polymerase activities could indicate either the presence of different active sites for distinct activities or the presence of a unique active site with different configurations depending upon the template used. Also, BI-RG-587 showed a mutually exclusive inhibition when combined with two other classes of HIV-1 RT inhibitors represented by phosphonoformic acid and 3'-azido-3'-dideoxythymidine triphosphate.


Cold resistance estimated on the basis of fetal hemoglobin changes during acute general cooling.

In order to find a criterion for human cold resistance and adaptability we studied the reaction of hematological indices to acute general cooling of the whole organism (12 degrees C, 1 h) in 60 male volunteers 19-25 years old living in Norilsk (69 degrees N) for about 2 1/2 years. The first group of subjects comprised of cold adapted individuals whose professional activity was associated with 4-5 hrs daily work outdoor in cold environment. The volunteers of the second group regularly worked indoor and are considered a priori cold unadapted. Our results indicate that fetal hemoglobin (HbF) concentration shows the most remarkable reaction to acute cooling in comparison with other parameters studied (erythrocyte count, hematocrit, total hemoglobin level). Cold exposure of unadapted young males was accompanied by a marked increase of HbF concentration in venous blood (from 1.27 +/- 0.13% to 2.04 +/- 0.02%, p less than 0.01), whereas the same procedure in the adapted subjects was followed by practically unchanged HbF levels. These findings lead us to the conclusion, that the change of the HbF level under acute general cooling can be considered a satisfactory criterion of human cold resistance.


Leukocyte traffic to sites of inflammation.

The adhesion of circulating leukocytes to the vascular endothelium is essential for effective host inflammatory and immune responses. Adhesion proteins expressed by both the leukocyte and endothelial cell have been well characterized, and studies of these molecules have shown that both cell types are actively involved in regulating these binding events. Most leukocyte (leukocyte integrins) and endothelial cell (vascular selectins, ICAM-1, and VCAM) adhesion proteins increase in expression and function in response to mediators released by inflamed tissues. In contrast, the expression and function of one type of leukocyte molecule, L-selectin (previously called LECAM-1, LAM-1, gp90MEL-14), is "down-regulated" by inflammatory signals. The purpose of this review is to summarize in vitro and in vivo regulatory and functional studies of some of the molecular mechanisms which regulate leukocyte-endothelial cell adhesion, with particular emphasis on L-selectin, and to present a hypothetical model of how these molecules may be orchestrated in vivo resulting in the control of host inflammatory responses.


Antigenic structure of the herpes simplex virus type 1 glycoprotein C: demonstration of a linear epitope situated in an environment of highly conformation-dependent epitopes.

A continuous epitope, situated within or in close proximity to antigenic site II of the herpes simplex virus type 1-specified glycoprotein C (gC-1), was identified. The continuous linear nature of the epitope, defined by a monoclonal antibody C2H12, was established by three independent lines of evidence: (i) The epitope was detectable by immunoblot under denaturing and reducing conditions. (ii) The epitope was detectable by RIPA of extracts from TM-treated HSV-infected cells, despite the malfolding caused by this treatment. (iii) The epitope was detected in an approximately 5,000-dalton papain fragment of gC-1. A mapping analysis, primarily based on use of mutant virus, expressing truncated gC-1 molecules, suggested that the mapping position of the epitope was delimited by amino acids 120 and 230. Other epitopes of this region of gC-1 are highly conformation-dependent, and the existence of a linear epitope, accessible on native gC-1, may facilitate the elucidation of the functional anatomy of gC-1.


[Inhibition of neovascularization and tumor growth by dexamethasone].

It has been proposed that angiogenesis inhibitors should be used for the treatment of diseases accompanied by an uncontrolled angiogenic response, particularly such disease occurring during progressive growth of solid tumors. In this study, the antiangiogenic effect of dexamethasone (DEX) was studied in a system using chorioallantoic membranes (CAM) of fertilized eggs and rabbit corneas. First, DEX was examined for its effect on embryonic angiogenesis using 4, 5 day old CAMs of chick embryos. After the shell and shell membrane was removed, an EV pellet with or without DEX was placed within a silicon ring. Two days later, the antiangiogenic response was evaluated by measuring the avascular zone of the CAM beneath the pellet. When the CAM showed an avascular zone of 3 mm or more in diameter, the response was scored as positive. DEX showed potent antiangiogenic activity and produced an avascular zone in 100% of CAMs at the highest dose tested. (250 ng/egg). Next, inhibitory effect of vascularization and tumor growth by local implant of DEX was observed in a rabbit cornea assay. An intra-corneal pocket extending to within 1 mm of the limbus was used to house a 1-mm3 piece of glioma. In the treatment group, DEX-containing EV pellet was inserted between the limbus and glioma. Ten days after the implantation of glioma and EV pellet, vascular response and tumor growth was evaluated. For morphologic studies, the excised corneas were fixed with formaldehyde fixative and sectioned for light microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)


Clinical pharmacology of mivacurium chloride: a review.

Mivacurium chloride (Mivacron) is a new benzylisoquinolinium choline-like diester neuromuscular blocking drug with an onset of action at equipotent doses that is comparable to atracurium and vecuronium but slower than succinylcholine. Its clinical duration (injection-25% recovery and injection-95% recovery) is twice that of succinylcholine but one-half to one-third that of atracurium and vecuronium. Mivacurium is easy to use as a continuous infusion and when used this way its recovery characteristics are unchanged. It is readily antagonized by anticholinesterase drugs. The ED95 in adults under narcotic-based anesthesia is 0.07-0.08 mg/kg. At twice the ED95 (0.15 mg/kg) onset time is about 2 to 3 minutes, duration to 25% recovery is 15 to 20 minutes, and 5-95% recovery time about 14 minutes. The mean infusion rate in adults is 6 micrograms/kg/min (range 2-15) with a 5-95% recovery time of 14 minutes. Enflurane and isoflurane require a 20-30% decrease in dosage; halothane, enflurane, and isoflurane prolong the duration of mivacurium 25-30%. The ED95 in children 2 to 12 years of age is slightly higher (0.09-0.11 mg/kg) with a faster onset and shorter duration. In these young patients, a dose of 0.2 mg/kg has an onset comparable to succinylcholine. Being chemically related to atracurium, mivacurium may cause histamine release. When administered rapidly at doses of 0.2 mg/kg or greater in adults, histamine release and transient hypotension have been observed. Doses of 0.2 mg/kg or higher are not recommended by the manufacturer. Mivacurium is metabolized by plasma cholinesterase. In vitro, the rate is about 70% that of succinylcholine. In patients with normal or slightly less than normal plasma cholinesterase activity, no prolonged durations of action have been observed. In patients heterozygous for the atypical gene and at a dose of 0.2 mg/kg, 50% prolongation has been shown. Those individuals homozygous for the atypical gene are exquisitely sensitive to mivacurium and have a markedly prolonged blockade that is readily reversible. In these patients and those with acquired deficiencies, mivacurium should not be used. The duration of action in elderly patients is comparable to that in the young, while in prerenal transplant patients, its duration is prolonged by about 50%, and in prehepatic transplant patients, duration of block is increased threefold. Mivacurium possesses the advantages of short duration, unchanged recovery characteristics following infusions (without phase II block or tachyphylaxis), and precise control.(ABSTRACT TRUNCATED AT 250 WORDS)


IL-1 production as a regulator of G-CSF and IL-6 production in CSF-producing cell lines.

We previously demonstrated that colony stimulating factor (CSF)-producing cell lines co-produce interleukin-1 (IL-1) and IL-6 in addition to CSFs. In the present study, we examined the role of IL-1 production in three human tumour cell lines producing granulocyte (G)-CSF, IL-1 and IL-6. Addition of anti-human IL-1 alpha antiserum to the culture caused a 90-62% reduction of G-CSF and a 85-44% reduction of IL-6 production, respectively, as evaluated by enzyme immunoassay in all three cell lines. The decrease of G-CSF and IL-6 production by the anti-IL-1 alpha antiserum was also confirmed at the level of mRNA expression. The anti-IL-1 alpha antiserum did not affect the growth of these cell lines. Excess recombinant IL-1 alpha exogenously added to the culture enhanced G-CSF and IL-6 production in all three cell lines. However, IL-1 alpha had little effect on the growth of these three cell lines. Neither anti-IL-6 nor anti-G-CSF antibodies affected the production of the other cytokines. These results indicate that IL-1 alpha regulates G-CSF and IL-6 production in these tumour cell lines, and suggest that the IL-1 production plays an important role in CSF-producing tumours.


Topology of the anion-selective porin Omp32 from Comamonas acidovorans.

Limited proteolysis experiments were performed with outer membranes from Comamonas acidovorans to probe the topology of its major protein component, the anion-selective porin Omp32. Proteinase K treatment above a critical temperature of 42 degrees C cleaved the surface-exposed regions of the porin, yielding membrane-embedded fragments which were separated by SDS polyacrylamide gel electrophoresis or reversed phase chromatography. The identification of the proteinase K-sensitive sites was performed by microsequencing. This allowed us to determine six surface-exposed sites of the porin, all located in nonconserved primary structure regions. These results along with the previously determined amino acid sequence and in conjunction with some structural constraints applicable to porins allowed us to propose a chain-folding model of the Omp32 porin. The features of our model are compared with the structure of the Rhodobacter capsulatus porin, recently established by X-ray crystallography (Weiss et al., 1991) and they are used to elucidate the structural basis of the anion selectivity.


Effectiveness of combinations of bispecific antibodies for delivering saporin to human acute T-cell lymphoblastic leukaemia cell lines via CD7 and CD38 as cellular target molecules.

We have investigated the effectiveness of three different F(ab' gamma)2 bispecific antibodies (BsAb) for delivering the ribosome inactivating protein (RIP) saporin via the CD7 or CD38 cell surface molecules to the human T-ALL cell lines HSB-2 and HPB-ALL. Inhibition of 3H-leucine uptake by target cells was used as the parameter of cellular cytotoxicity. Used singly against HSB-2 cells in the presence of varied concentrations of saporin, an anti-CD7 BsAb, (HB2 x DB7-18) and an anti-CD38 BsAb (OKT10 x RabSap), gave 435- and 286-fold increases in saporin toxicity, respectively. For HPB-ALL cells the anti-CD7 BsAb performed poorly giving only an eight-fold increase in toxicity whilst on the same cell line the anti-CD38 BsAb was highly potent giving an 80,000-fold increase in saporin toxicity. A combination of both BsAb used together against HSB-2 cells was ten times more effective, than the best single BsAb HB2 x DB7-18 used alone. Kinetic studies conducted with HSB-2 cells revealed that the BsAb combination also gave an increased rate of protein synthesis inactivation in comparison to either BsAb used alone. These investigations clearly demonstrate a synergistic action when both BsAb are used in combination to target saporin against CD7 and CD38 expressed on the surface of the HSB-2 cell line.


Localization of interferon-induced lupus inclusions demonstrated by computer image reconstruction of monensin-treated Daudi cells.

A structural analysis of cells that contained the interferon-alpha-induced lupus inclusions (LI) was performed using a high-voltage electron microscope to determine the exact cellular location of LI and their association with normal cell organelles. LI were induced in the human B lymphoblastoid cell line, Daudi, by culturing with the pure recombinant human leukocyte interferon, IFLrA. Just prior to harvesting, a portion of the cells was treated with monensin to selectively swell the Golgi apparatus, and thereby simplify their identification using the electron microscope. Organellar associations between LI and the outer nuclear envelope and Golgi apparatus were identified in stereopairs of 1-micron sections prepared from both cells that were not treated with monensin and those that were treated with monensin. Serial 0.25-micron sections of the monensin-treated cells were prepared, and seven arbitrarily chosen cells were examined. Each of these cells contained a single LI, and it formed throughout an endoplasmic-reticulum region that made contact with both the outer nuclear envelope and the Golgi vesicles. Reconstruction of a cell by computer from the digitized negatives of serial sections clearly illustrated these relationships. This study reports the first determination of the association between LI and the Golgi apparatus. It also identifies the presence of only one LI in every cell, and the routine association of the LI with both the outer nuclear envelope and the Golgi apparatus. The unique cell location of LI formation suggests their functioning in membrane biogenesis, the trafficking of proteins to the plasma membrane or to cytoplasmic vesicles, or the processing of proteins for secretion.


Monoclonal antibody FW6 generated against a mucin-carbohydrate of human amniotic fluid recognises a colonic tumour-associated epitope.

Mucus glycoproteins of human amniotic fluid were used to generate a monoclonal antibody (MAb) FW6, which stained the intestine of a 24 week stage fetus. In adults, 76% of colonic adenocarcinomas (13/17) showed strong expression of the FW6 epitope, but it was not detected in the histologically normal mucosae adjacent to the tumours or in normal left colon mucosa. In addition, MAb FW6 stained large cell carcinomas of the lung (2/3), gastric carcinomas (5/11), and ovary adenocarcinomas (3/4). The expression in carcinomas can also be called ectopic for testing normal tissues. MAb FW6 was also reactive with pyloric mucus glands, Brunner's glands of the duodenum, Paneth cells of the ileum, pancreatic ducts, absorptive cells of the right colon, bronchiolar glands, kidney urothelia, and with a restricted number of normal mucinous tubuli of salivary gland. It was demonstrated to be under the control of the secretion gene only in intestinal Paneth cells and absorptive cells of the right colon. Comparative histochemical analysis comprising a panel of MAbs suggests that the corresponding epitope of the MAb FW6 is a type II chain related carbohydrate structure belonging to the Lex/Ley-antigen family, but is different from short chain Lex and Ley.


Monoclonal antibodies in the detection of bone marrow metastases in small cell lung cancer.

Using conventional examination (CE) of H&E stained slides from bone marrow aspirates, metastases can be detected in approximately 25% of patients with small cell lung cancer. We investigated a panel of monoclonal antibodies using immunohistochemistry in the diagnosis of bone marrow infiltration from SCLC and compared the results with CE. Seven monoclonal antibodies raised against epithelial antigens (CAM 5.2, MOV 15, NCCST 433, PE 35, LCA1/L38, HMFG 1 AND HMFG 2) were applied on bone marrow sections from three groups of patients (pts): (1) 19 pts in whom SCLC-metastases were detected by CE, (2) 44 pts with SCLC in whom metastases could not be detected by CE, and (3) 20 pts with non-malignant bone marrow diseases. All the antibodies except LCA1/L38 were positive in 60-90% of the slides with infiltrating tumour cells in group 1. No positive tumour cells were detected in group 2. A few plasma cells and megakaryocytes were slightly positive for MOV 15 and NCCST 433, but no other positive cells were detected in group 3. In conclusion, the monoclonal antibodies used in this study may be useful for diagnostic purposes when a suspicious looking infiltration is detected by CE. However, these antibodies could not detect metastatic tumour cells in the bone marrow sections from patients in whom CE did not reveal any tumour cells.


A model of glucocorticoid receptor unfolding and stabilization by a heat shock protein complex.

It has recently been reported that incubation of avian progesterone receptors, mouse glucocorticoid receptors, or the viral tyrosine kinase pp60src with rabbit reticulocyte lysate reconstitutes their association with the 90 kDa heat shock protein, hsp90. The reassociation is thought to require unfolding of the steroid receptor or pp60src before hsp90 can bind. The unfoldase activity may be provided by hsp70, which is also present in the reconstituted receptor heterocomplex. In this paper we review evidence that hsp70 and hsp90 are associated in cytosolic heterocomplexes that contain a limited number of other proteins. From an analysis of known receptor-hsp interactions and a predicted direct interaction between hsp90 and hsp70 we have developed an admittedly very speculative model of glucocorticoid receptor unfolding and stabilization. One important feature of the model is that the receptor becomes attached to a heat shock protein heterocomplex rather than undergoing independent unfolding and stabilization events. The model requires that hsp70 and hsp90 bind directly to the receptor at independent sites. Importantly, the model accommodates the stoichiometry of 2 hsp90 per 1 molecule of receptor that has been assayed in the untransformed GR heterocomplex in cytosols prepared from hormone-free cells.


Domain structure and antiparallel dimers of microtubule-associated protein 2 (MAP2).

We have studied the microtubule-associated protein MAP2 from porcine brain and its subfragments by limited proteolysis, antibody labeling, and electron microscopy. Two major chymotryptic fragments start at lys 1528 and arg 1664, generating microtubule-binding fragments of Mr 36 kDa (303 residues, analogous to the "assembly domain" of Vallee, 1980) and 18 kDa (167 residues). These fragments can be labeled with the antibody 2-4 which recognizes the last internal repeat of MAP2 (Dingus et al., 1991). The epitope of another monoclonal antibody, AP18 (Binder et al., 1986), was mapped to the first 151 residues of MAP2. The interaction with AP18 is phosphorylation dependent; dephosphorylated MAP2 is not recognized. Intact MAP2 forms rod-like particles of 97 nm mean length, similar to Gottlieb and Murphy's (1985) observations. Both antibodies bind near an end of the rod, suggesting that the sequence and the structure are approximately colinear. There is a pronounced tendency for MAP2 to form dimers whose components are nearly in register but of opposite polarity. MAP2 can also fold in a hairpin-like fashion, generating 50-nm rods, and it can self-associate into oligomers and fibers. The 36-kDa microtubule-binding fragment also has a rod-like shape; its mean length is 49 nm, half of the intact molecule, even though the fragment contains only one-sixth of the mass. The antibody 2-4 decorates one end of the rod, similar to the intact protein. The fragment also forms antiparallel dimers, but its tendency for higher self-assembly forms is much lower than with intact MAP2.


Steroids inversely affect the biosynthesis and secretion of human prostatic acid phosphatase and prostate-specific antigen in the LNCaP cell line.

In order to elucidate the mechanism of androgen-regulation of genes expressed only in the prostate gland, the effects of steroid hormones on the biosynthesis and secretion of human prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) were studied in the human prostatic carcinoma cell line, LNCaP. This cell line produces PAP and PSA, both of which were found to be similar to the proteins purified from and located in human prostatic tissue, as shown by Western blot analysis. The synthetic androgen, R1881, regulated the biosynthesis of these two important tumour marker proteins inversely: the amount of PSA released into the medium was increased to 506% +/- 100 of the control levels, while that of PAP was decreased to 26% +/- 3, in 7 days. These effects were dependent on the concentration of the steroid in the growth medium. The androgen-dependent changes observed in the amounts of the secreted proteins were correlated with alterations in their intra-cellular levels. LNCaP cells were found to have very different capacities for secreting PAP and PSA. Whereas the measurable, cellular amounts of PSA and PAP were of similar magnitudes, much larger amounts of PSA than PAP were secreted into the medium. PSA was also found to be more stable than PAP in the culture medium of the LNCaP cells. Other steroids could elicit effects on PAP and PSA biosynthesis similar to those induced by R1881, and the combined effects of effective concentrations of these steroids were undistinguishable from those caused by each one of them separately, suggesting that all these compounds compete for binding to the same modified androgen receptors of the LNCaP cells. Thus, our results confirm the observations of the altered nature of the LNCaP androgen receptors, and demonstrate the ability of these ligands to produce changes in the expression of androgen-dependent prostatic genes. The fact that the changes observed at the protein level were accompanied by increased levels of PSA mRNAs and by decreased levels of PAP mRNA in steroid-treated cells, suggests that one of the targets of androgen and steroid action in the regulation of these genes is at the mRNA level.


Regulation of tumor-host interactions in breast cancer.

Breast cancer has a striking dependence upon steroid and other endocrine hormones in its onset, regulation, and malignant progression to its most deadly forms. The epithelium of the normal mammary gland is also regulated by the ovarian endocrine steroids estrogen and progesterone, by other endocrine hormones, and by poorly defined influences of the stromal cells and basement membrane. The onset and development of cancer appears to involve tumor misinterpretation of and/or desensitization to host regulatory signals, and finally to releasing its own hormonal signal to reorganize the host for its own benefit. Current studies are beginning to examine mediators of tumor-host interaction and their regulation by steroid hormones. Important tumor-host interactions under investigation include desmoplasia, angiogenesis, metastases and immunosuppression.


Calcium signaling and secretory responses in agonist-stimulated pituitary gonadotrophs.

In cultured pituitary gonadotrophs, gonadotropin-releasing hormone (GnRH) caused dose-dependent and biphasic increases in cytoplasmic calcium concentration ([Ca2+]i) and LH release. Both extra- and intracellular calcium pools participate in GnRH-induced elevation of [Ca2+]i and LH secretion. The spike phase of the [Ca2+]i response represents the primary signal derived predominantly from the rapid mobilization of intracellular Ca2+. In contrast, the prolonged phase of the Ca2+ signal depends exclusively on Ca2+ entry from the extracellular pool. The influx of Ca2+ occurs partially through dihydropyridine-sensitive calcium channels. Both [Ca2+]i and LH responses to increasing concentrations of GnRH occur over very similar time scales, suggesting that increasing degrees of receptor occupancy are transduced into amplitude-modulated Ca2+ responses, which in turn activate exocytosis in a linear manner. However, several lines of evidence indicated the complexity over the relationship between Ca2+ signaling and LH exocytosis. In contrast to [Ca2+]i measurements in cell suspension, single cell Ca2+ measurements revealed the existence of a more complicated pattern of Ca2+ response to GnRH, with a biphasic response to high agonist doses and prominent oscillatory responses to lower GnRH concentrations, with a log-linear correlation between GnRH dose and the frequency of Ca2+ spiking. In addition, analysis of the magnitudes of the [Ca2+]i and LH responses of gonadotrophs to a wide range of GnRH concentrations in the presence and absence of extracellular Ca2+, and to K+ and phorbol ester stimulation, showed non-linearity between these parameters with amplification of [Ca2+]i-mediated exocytosis. Studies on cell depleted of protein kinase C under conditions that did not change the LH pool suggested the participation of protein kinase C in this amplification, especially during the plateau phase of the secretory response to GnRH.


Transcriptional activation of jun and actin genes by estrogen during mitogenic stimulation of rat uterine cells.

Estrogens induce transcriptional activation of c-fos and c-myc proto-oncogenes during mitogenic stimulation of human, chicken, mouse and rat cells in vivo and in vitro. In this paper we show that 17 beta-estradiol injected into adult ovariectomized rats increases c-jun, jun-B and jun-D gene transcription in the uterus. Kinetics and amplitude of response are different for each gene, since c-jun is activated first, within 30 min after injection, followed by jun-D and jun-B, 60 and 90 min after injection, respectively. Maximal activation of jun-B marks a drop in transcription of all the jun genes. Furthermore, transcriptional activation by 17 beta-estradiol of the growth-regulated beta- and gamma-cytoskeletal actin genes is prevented by an inhibitor of protein synthesis, indicating that it is a secondary response to the hormone. These data support the hypothesis that during growth stimulation of target cells the estrogen receptor induces transcription of regulatory genes, triggering in this way a cascade of gene regulation events that results in progression through the cell cycle.


Expression of estrogen receptor in the rat brain: detection of its mRNA using reverse transcription-polymerase chain reaction.

Reverse transcription-polymerase chain reaction (RT-PCR)-Southern blotting was employed for biochemical detection and measurement of the estrogen receptor messenger ribonucleic acid (ERmRNA) in various parts of the Wistar strain female rat brain. Total RNA extracted from the tissues was subjected to RT-PCR using the primer set which flanked the part(287bp) of the estrogen binding domain-corresponding region of the rat ERcDNA. It was confirmed that the RT-PCR product corresponded to the part of the ERcDNA by direct nucleotide sequencing of the product. Moreover, the level of ERmRNA could be determined by Southern blotting which was highly sensitive and produced quantitative results. The RT-PCR product of about 290 bp, corresponding in length to the distance between two primers, was generated from RNA of all the tissues examined. The levels of the product were as follows; anterior hypophysis greater than hypothalamus and preoptic area, amygdala much greater than cerebral cortex, cerebellum. These results indicate that ERmRNA is widely distributed in the whole brain, implying some physiological action of estrogen on target and 'non-target' brain regions.


Expression of progesterone receptor in the neonatal rat brain cortex: detection of its mRNA using reverse transcription-polymerase chain reaction.

Our previous reports have revealed the postnatal developmental pattern of progesterone receptor (PR) in the rat cerebral cortex, but little is known about PRmRNA changes in the tissue so far. In the present study, we have attempted to detect and quantify PRmRNA in the tissue by the use of the reverse transcription-polymerase chain reaction (RT-PCR)-dot blot analysis. Cerebral cortical tissues were dissected from female Wistar rats at 2 and 8 days of age. Total RNA extracted from the tissues was reverse transcribed, followed by PCR. The PCR primer set, whose sequence was derived from the human clone, flanked the part (320 bp) of the human PRcDNA which corresponded to the progesterone binding domain. The 320 bp of the RT-PCR product was generated from rat uterine RNA. The amino acid sequence deduced from the nucleotide sequence of the product had a 95.5% identity with the corresponding region in human PR, confirming that the product had originated from the rat PRmRNA. Moreover, a highly sensitive and quantitative assay for rat PRmRNA had been developed by the use of RT-PCR-dot blotting. The PRmRNA was detectable in the cerebral cortex of both 2- and 8-day-old rats by the assay. Furthermore, the level of cortical PRmRNA of the 8-day-old rats were greater than the 2-day-old rats. These results indicate that the RT-PCR assay is useful for detection and quantification of PRmRNA in the neonatal rat cerebral cortex, and that increment of the cortical PRmRNA in the 8-day-old rat is associated with a drastic change of the level of the cortical PR around day 10.


Immunoreactive EGF in human benign prostatic hyperplasia: relationships with androgen and estrogen receptors.

Benign prostatic hyperplasia (BPH) is a sex steroid dependent disease. Estrogens and androgens can modulate in different mammalian tissues epidermal growth factor (EGF) production and/or secretion. In order to clarify the relationships between estrogen and androgen receptor concentrations and those of immunoreactive EGF (irEGF), we have evaluated these parameters in 14 human BPH samples, by means of a dextran-coated charcoal method and radioimmunoassay, respectively. Cytosolic steroid receptors did not seem to correlate with irEGF. A linear significative relationship was evident between nuclear androgen receptor (ARn) levels and endogenous irEGF but not between nuclear estrogen receptors and irEGF: in ARn negative BPH samples, irEGF levels were lower than in ARn positive ones. Therefore, it is possible that androgens act at prostatic tissue level, through their own receptors, by modulating EGF production and/or secretion.


Partial purification of 3 alpha- and 3 beta-hydroxysteroid dehydrogenases from human hyperplastic prostate. Comparison between the two enzymes.

3 alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) activity has been purified to homogeneity, the enzyme is a monomer with a Mw of 32,000 Da. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) activity has been partially purified and has an apparent Mw of 30,000 Da. Both enzymes have the same cofactor requirements, optimal pH. However, 3 beta-HSD appeared to be an integral protein dependent on protein environment for its activity while 3 alpha-HSD activity is a protein more loosely associated to membranes.


17 beta-acylurea derivatives of 4-azasteroids as inhibitors of testosterone 5 alpha-reductase.

A series of 17 beta-acylurea-4-aza-5 alpha-androstan-3-one derivatives has been assayed in vitro as inhibitors of testosterone 5 alpha-reductase, using the particulate fraction of human hyperplastic prostate and rat prostate as enzyme sources. The most active derivatives were 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-dicyclohexylurea (compound 1) and 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-diisopropylurea (compound 3) which demonstrated IC50 values of 41 and 55 nM for the human enzyme and of 83 and 53 nM for the rat enzyme, respectively. Neither compound showed any relevant binding affinity to the rat prostate androgen receptor (IC50 of approximately 100 and 84 microM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, compound 3 (laboratory code FCE 26073), at 3 mg/kg/day, significantly decreased the ventral prostate growth promoting effect of TP by 40-50%, whereas compound 1 was ineffective up to the dose of 10 mg/kg/day.


Influence of captopril on adrenal cytochrome P-450s and adrenodoxin expression in high potassium or low sodium intake.

To investigate the role of the renin-angiotensin system in steroidogenic enzyme expression, the angiotensin-I converting enzyme inhibitor captopril was administered in conjunction with high potassium (K+) or low (Na+) intake to rats for a 7-day period. Northern blot analysis of adrenocortical zona glomerulosa RNA revealed that sodium restriction markedly increased mRNA production of P-450scc (3.1-fold) and P-450(11 beta) (3.4-fold) as well as of the electron donor adrenodoxin (2.0-fold). Captopril combined to the low Na+ diet led to suppression of these effects and, as also seen with captopril alone, further diminished P-450(11 beta) mRNA levels below controls. These responses were accompanied by parallel changes in respective protein levels of the enzymes as indicated by Western blot analyses. Captopril was also shown to inhibit the K(+)-stimulated levels of P-450(11 beta) mRNA (3.3-fold) and protein (1.4-fold) beneath control values (0.6- and 0.8-fold, respectively). On the other hand, increased P-450scc mRNA and protein levels by K+ loading were not affected by captopril treatment. No response was observed in any steroidogenic enzyme expression in zona fasciculata-reticularis following either diet with or without captopril. Thus, the inhibitory effect of captopril on stimulated steroidogenesis seemed to be mediated in part through transcriptional regulation of P450s. In addition, it appeared that P-450(11 beta) expression might be under the control of the renin-angiotensin system in both high K+ and low Na+ diets as opposed to the K+ stimulation of P-450scc where other mechanisms seemed to be involved.


The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin.

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 +/- 1.9 ng and 8.0 +/- 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K+ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K+. AngiotensinII (AII) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10(-8) M AII, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10(-8) M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of AII and ACTH on adrenal cytochrome P-450(11 beta) involved in the last steps of aldosterone formation were evaluated by combined in vivo and in vitro experiments. The P-450(11 beta) mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and AII differentially regulate P-450(11 beta). It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.


Electrophysiological recordings in solitary photoreceptors from the retina of squid, Loligo pealei.

A protocol was developed to isolate enzymatically photoreceptors from the retina of the squid, Loligo pealei. The procedure routinely results in a high yield of intact cells. Examination of solitary photoreceptors under Nomarski optics revealed that the fine morphological features described in anatomical studies of retinal sections are retained. The distal segment is up to 250 microns long, 4-7 microns wide, covered in part by short microvilli; the inner segment and the cell body, with the initial portion of the axon, are also clearly discernible in solitary cells. Suction electrode measurements performed from the cell body confirmed that responsiveness to light survived cell isolation. Macroscopic membrane currents were measured using the whole-cell tight-seal technique, and the perforated-patch method. Step depolarizations of membrane voltage administered in the dark elicited a slowly activating, sustained outward current. Light stimulation evoked an inward current graded with stimulus intensity; the peak current could amply exceed 1000 pA. Intense photostimulation gave rise to a prolonged inward aftercurrent that lasted for tens of seconds. On-cell patch recording along the intermediate segment and most of the smooth areas of the distal segment showed a large incidence of silent patches, with the occasional presence of voltage-dependent channels. On the other hand, channel activity could be recorded more frequently from electrode placements near the apical tip of the cell, where the presence of microvilli could be confirmed visually. Some patches were unresponsive to voltage stimulation applied in the dark but produced distinct bursts of channel openings after illumination. The feasibility of single-cell electrophysiology in isolated photoreceptors, together with the growing body of biochemical information on cephalopod preparations, makes squid an attractive model system to investigate the visual process in invertebrates using multiple experimental approaches.


The 11q23 breakpoint in acute leukemia with t(11;19)(q23;p13) is distal to those of t(4;11), t(6;11) and t(9;11).

Thirteen cosmid probes were mapped on the long arm of chromosome 11 between 11q22 and 11q24 by nonradioactive in situ hybridization. Starting with these localizations and those of other probes mapped to 11q23, four acute leukemias with translocations involving 11q23 were studied with the same method. The translocation breakpoints of the t(4;11)(q21;q23), t(6;11)(q27;q23), t(9;11)(p21-p22;q23), and t(11;19)(q23;p13) were confirmed to be distal to CD3D. The probe cC111-304 was proximal to the t(11;19) breakpoint while distal to the breakpoints of the other rearrangements. In view of the diversity of chromosomal abnormalities involving band 11q23, our finding extends the molecular heterogeneity of the breakpoint localization in leukemias with rearrangements involving 11q23.


Genetic alterations of the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q in human breast carcinomas.

Fifty-nine primary breast carcinomas and 11 metastases were examined to identify genetic alterations in the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q. Loss of heterozygosity (LOH) was frequently observed on chromosome arms 17p (p144D6 lost in 75%, pYNZ22.1 in 55%, and TP53 in 48% of the primary tumours), 13q (RBI lost in 40% of the primary tumours), and 17q (pRMU3 lost in 35%, pTHH59 in 29%, and NM23HI in 26% of the primary tumours). Loss of all the markers except p144D6 was observed even more frequently in the metastases. Pairwise comparisons for concordance of allele losses on 17p indicated that there might be two genes on 17p implicated in breast cancer development; the TP53 gene and a gene located close to the p144D6 and pYNZ22.1 markers. LOH of the RBI gene was associated with LOH of pYNZ22.1 and p144D6, but not with LOH of TP53. LOH of RBI and TP53 was associated with occurrence of ductal carcinomas, RBI and p144D6 losses with tumour size, and p144D6 losses with positive node status as well. LOH of TP53 and the three 17q markers NM23HI, pTHH59, and pRMU3 was most frequently observed in tumours from postmenopausal women. p144D6 losses occurred most frequently in progesterone receptor-negative tumours, whereas pTHH59 losses occurred most frequently in oestrogen receptor-negative tumours. LOH of the investigated loci was not associated with ERBB2 protooncogene amplification, with positive family history of breast cancer, or with survival.


Reciprocal translocation t(12;22)(q13;q13) in clear-cell sarcoma of tendons and aponeuroses.

Clear-cell sarcoma is a rare soft tissue sarcoma that displays certain similarities to malignant melanoma. In this paper we describe the karyotypic findings and in vitro growth characteristics of a short-term-cultured clear-cell sarcoma. The cultured tumor cells had preserved immunohistochemical characteristics and certain ultrastructural features of the primary tumour, including positivity for vimentin, S-100 protein, and a melanoma-associated antigen, supporting the authenticity of the cultured cells. Cytogenetic analysis revealed an abnormal stemline karyotype of 49,XY, -1, +8, +8, +12, +der(1)t(1;?)(p36.1-.3;?), t(12;22)(q13;q13). A similar or identical t(12;22) was recently reported in two of four clear-cell sarcomas. It is suggested that the t(12;22)(q13;q13) is a primary cytogenetic abnormality in clear-cell sarcoma and distinguishes this tumor type from malignant melanoma.


Nonradioactive in situ hybridisation of the translocation t(1;7) in myeloid malignancies.

Bone marrow cells of four patients with t(1;7) and myelodysplasia or acute myeloid leukemia were analyzed using nonradioactive in situ hydridisation. As probes, centromeric alphoid DNA sequences of chromosomes 1 and 7, a satellite DNA probe for 1q12, and chromosome-specific libraries of chromosomes 1 and 7 were used. The breakpoints of the t(1;7)(p11;p11) as determined by banding analysis could be studied more accurately, and the recently proposed designation t(1;7)(cen;cen) was confirmed in all four cases. Colocalization of alphoid DNA sequences of chromosomes 1 and 7 by double target in situ hybridisation was demonstrated in metaphase cells and also in interphase nuclei. The in situ hybridisation method described is applicable for the screening of peripheral blood cells or archival material.


Chromosomal in situ suppression hybridization of immunologically classified mitotic cells in hematologic malignancies.

Chromosomal in situ suppression (CISS) hybridization was performed with library DNA from sorted human chromosomes 8, 9, 15, 17, 21, and 22 on immunologically stained bone marrow cells of four patients with a hematologic neoplasm, including two patients with myelodysplastic syndrome and trisomy 8, one patient with promyelocytic leukemia bearing the translocation t(15;17)(q22;q11-12), and one patient with chronic myeloid leukemia and the translocation t(9;22)(q34;q11). In all patients, the results of conventional karyotype analysis could be confirmed by one- or two-color CISS hybridization using the appropriate chromosome-specific libraries. Our results show that CISS hybridization can detect both numerical and structural chromosome changes in immunologically classified cells with high specificity and reliability. The fact that chromosome spreads of very poor quality can now be included in such analyses is a decisive advantage of this approach. In addition, the suitability of this approach for interphase cytogenetics is discussed.


Chromosomal abnormalities in skin following total body or total lymphoid irradiation.

Patients undergoing bone marrow transplantation often receive total body or total lymphoid irradiation as part of the conditioning regimen prior to marrow infusion. The cytogenetic effects of this therapy on skin fibroblasts were studied. Fibroblast cultures from eight skin biopsies were harvested in early passages for G-banded chromosome analysis. Four biopsies were from three patients who had high-dose cyclophosphamide and total body radiotherapy; one was from within and one was from outside the radiation field of a patient who had high-dose cyclophosphamide and lymphoid radiotherapy, one was from a patient who had combination chemotherapy alone, and one was from a normal control. No abnormal mitoses were found in the control or the patient who had chemotherapy alone, and only two of 30 mitoses from skin outside the lymphoid radiotherapy field were abnormal. However, most cells (49-88%) from five biopsies within radiotherapy fields were abnormal. Typically, abnormal karyotypes were pseudodiploid and contained multiple balanced rearrangements, of which reciprocal translocations were most common. The data indicate that the radiotherapy used for bone marrow transplantation induces extensive, sustained chromosome abnormalities in vivo in skin fibroblasts.


Cytogenetic abnormalities in an ossifying fibroma from a patient with bilateral retinoblastoma.

Cytogenetic analysis of a cemento-ossifying fibroma from a patient with nonfamilial bilateral multicentric retinoblastoma revealed three reciprocal translocations with the karyotype 46,XY,t(1;18)(q21;q21.3),t(3;10)(p13;q22),t(6;11)(p22;p15). Routine and high-resolution cytogenetic analysis of peripheral blood leukocytes showed an apparently normal, 46,XY chromosome pattern with no deletion of chromosome 13. Molecular analysis demonstrated no gross differences in the retinoblastoma gene or the TP53 gene between constitutional and tumor DNA. This is the first cytogenetic analysis of a cemento-ossifying fibroma and the first report of this tumor in a retinoblastoma patient. The data may be added to the small, but growing literature on cytogenetic aberrations in benign tumors and may lend insight into genes involved in cell proliferation and neoplastic transformation.


t(11;18)(q21;q21) is a recurrent chromosome abnormality in small lymphocytic lymphoma.

We have identified two cases of previously untreated, small lymphocytic lymphoma with extranodal involvement, which had a reciprocal translocation, t(11;18)(q21;q21), as the sole cytogenetic abnormality. These two cases are remarkably similar to two previously reported cases carrying this translocation with regard to clinical features, cytogenetic abnormality, histologic subtype, and immunophenotype. Molecular genetic analysis of these two cases revealed clonal gene rearrangement of the IGH locus but only germline configuration of the BCL2 oncogene at 18q21 when probes and conditions that usually identify BCL2 rearrangement in lymphomas were used. Lymphomas bearing an (11;18) rearrangement appear to make up a phenotypically identifiable subgroup. Identification of the genes at the translocation breakpoints will be important.


Losses of 3p, 11p, and 13q in EJ/ras-transformable simian virus 40-immortalized human uroepithelial cells.

Five independent clones of Simian virus 40 (SV40)-immortalized human uroepithelial cells (CK/SV-HUC) were established after transfection of HUC cultures from the same tissue donor with plasmids encoding SV40 large T and small t antigen genes. Each CK/SV-HUC clone contained a unique SV40 integration site, and all expressed similar levels of SV40 mRNA. All five clones were nontumorigenic, but clones 2, 4, and 5 tumorigenically transformed after transfection at P19 with mutant EJ/ras and also spontaneously after 40 serial passages in vitro. In contrast, CK/SV-HUC clones 1 and 3 did not transform when either approach was used. These differences in transformability among CK/SV-HUC clones could not be predicted based on differences in SV40 gene expression nor on any in vitro growth property tested. In cytogenetic analyses, a transformable clone showed losses of three chromosome arms containing putative cancer suppressor gene regions, including 3p14----pter, 13q, and 11p, whereas the nontransformable clones showed none of these losses. Thus these data indicate that genetic losses on 3p, 11p, and 13q may contribute to tumorigenic transformation of SV40-immortalized human uroepithelial cells.


Amplification of the topoisomerase II alpha gene in a non-small cell lung cancer cell line and characterisation of polymorphisms at the human topoisomerase II alpha and beta loci in normal tissue.

DNA was prepared from normal tissue and 19 lung cancer cell lines. Using probes which detect restriction fragment length polymorphisms at both the topoisomerase II alpha and beta loci, heterozygosity was detected at a frequency of 0.17 and 0.37 for the alpha and beta loci, respectively. Southern blot analysis of DNA extracted from lung cancer cell lines detected amplification of both the topoisomerase II alpha and ERBB2 genes in the adenocarcinoma line Calu3. These results indicate that topoisomerase II alpha and ERBB2 may be closely linked on chromosome 17 and coamplified during adenocarcinoma progression. Since topoisomerase II is a target for several anticancer drugs, it will be of interest to study alterations to topoisomerase II genes during tumour development, as these may in part determine the response of the tumour to chemotherapy.


Abnormalities of chromosomes 7 and 22 in human malignant pleural mesothelioma: correlation between Southern blot and cytogenetic analyses.

Southern blot hybridization studies were performed on 23 mesothelioma primary tumor specimens to detect chromosome 1-, 7-, and 22-specific numerical changes, gene amplifications, and gene rearrangements. The molecular findings were compared with previous cytogenetic findings. No gene rearrangements or amplifications were detected. A numerical abnormality of chromosome 7 was detected by Southern blot analysis in two cases in which no numerical abnormality had been detected by the previous chromosome study. A numerical aberration of chromosome 22 was detected in five cases, which was compatible with the cytogenetic finding of monosomy 22 in these cases. The Southern blot results for the copy numbers of chromosomes 7 and 22 were concordant with the cytogenetic findings in 65%-80% of the cases.


t(11;18)(q21;q21.1): a recurring translocation in lymphomas of mucosa-associated lymphoid tissue (MALT)?

A distinct subtype of extranodal malignant lymphoma derived from mucosa-associated lymphoid tissues (MALT) has recently been defined. We have detected an acquired t(11;18)(q21;q21.1) in a patient with a MALT lymphoma of the stomach. This translocation has previously been described in two other patients with extranodal lymphoma. The BCL2 oncogene, which is located at band 18q21.3 and is activated by the t(14;18)(q32;q21) in follicular lymphoma, was not rearranged in this case. This newly identified t(11;18) may be a recurring translocation specifically associated with MALT lymphomas. Genes located at the breakpoint sites of chromosome 11 and/or chromosome 18 may be crucial to the pathogenesis of this type of malignant lymphoma.


Molecular cytogenetic analysis of a complex t(10;22;11) translocation in Ewing's sarcoma.

Fluorescence in situ hybridization (FISH) using chromosome-specific plasmid libraries and chromosome region-specific DNA markers allowed the characterization of a t(10;22;11) (p11.2;q12;q24) in a Ewing's sarcoma (ES). This study illustrates the usefulness of molecular cytogenetic analysis of ES, especially for determining the localization of the translocated 11q24-25 segment in complex or variant translocations.


A synovial sarcoma with t(X;18)(p11;q11) in a patient with Turner's syndrome.

The first case of synovial sarcoma in a patient with Turner's syndrome (45,X) is reported. Cytogenetic analysis of the tumor cells showed that the only X chromosome was involved in the t(X;18)(p11;q11) characteristic of synovial sarcoma.


Changes at the N-terminus of human brain creatine kinase during a transition between inactive folding intermediate and active enzyme.

CK-STAR, a monoclonal antibody against human brain creatine kinase (CK), can be shown by chemical cleavage mapping and peptide synthesis to recognize an epitope at the free N-terminus of the enzyme. The epitope could be largely reproduced by a synthetic peptide based on the first 18 amino acids and could be partly formed by the first 11 amino acids. The antibody did not bind to native CK, but it did bind to CK in various partially denatured forms and to an enzymically inactive intermediate in the refolding process. Competitive binding studies have shown that the N-terminal conformations of both the refolding intermediate and the free peptide resemble that of CK partially denatured by attachment to plastic. The results suggest that the final stages of CK refolding and reactivation involve a structural change at the N-terminus or its interaction with some other part of the CK molecule, thus masking the CK-STAR epitope.


Human alpha 2-HS-glycoprotein/bovine fetuin homologue in mice: identification and developmental regulation of the gene.

Human alpha 2-HS-glycoprotein (AHSG) is a plasma protein synthesized in liver and selectively concentrated in bone matrix. It has been reported to be involved in bone formation and resorption as well as immune responses. Recently, AHSG was found to be the species equivalent protein of fetuin, the major fetal serum protein in cattle and sheep. The function and regulation of AHSG/fetuin in different species are not understood. We have isolated a liver cDNA clone that encodes the human AHSG/bovine fetuin homologue in the mouse. The AHSG/fetuin gene may have a role in differentiation since it is expressed in mouse limb buds and brain only at certain stages during development. Mouse liver AHSG/fetuin mRNA was present at low level at 12 days gestation but its level increased during the late part of gestation and peaked between 1 to 3 months after birth. The regulation of mouse AHSG/fetuin synthesis during development was found to be significantly different from that of sheep and bovine fetuin. Compared to fetuin, which is reduced in adult to 1 to 2% of the fetal level, mouse AHSG synthesis subsides only 50% 4 months after birth.


spoVG sequence of Bacillus megaterium and Bacillus subtilis.

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases.


The control of mRNA stability in Escherichia coli: manipulation of the degradation pathway of the polycistronic atp mRNA.

The physical and functional stabilities of genes in the atp operon fall into two classes. The first two genes, atpI and atpB, are rapidly inactivated and degraded at the mRNA level. The remaining seven genes are more stable. In order to investigate how these stabilities are determined, DNA sequences encoding mRNA structures that influence degradative events in other systems, including RNAse III sites and REP sequences, were subcloned or synthesized and inserted into non-coding regions of the operon. The effects of insertion of an RNAse III site depended on whether cleavage left an unstable 3' end or a stabilizing stem-loop upstream of the cutting point. Generation of an unstable 3' end destabilized the neighbouring upstream atp gene, thus modifying the course and rate control of degradation. Removal of the atp transcriptional terminator attenuated expression of the last gene of the operon, atpC. This effect was reversed by substitution of an alternative stem-loop for the terminator. REP sequences inserted into intercistronic regions apparently could not influence rate-controlling steps. The reported data shed light on the factors controlling the inactivation and degradation of genes in the polycistronic atp mRNA, and are discussed in relation to the general role of degradation processes in the control of gene expression.


Alpha-fetoprotein expression in fetal kidney cells does not require enhancers.

Expression of the alpha-fetoprotein (AFP) and albumin genes was examined in fetal mouse kidney by analysis of tissue mRNA pool sizes during development and transient expression assays in primary kidney tissue culture cells. AFP is expressed at a much lower level in kidney than in liver but transcription of the gene is activated early during development and repressed after birth with a time-course similar to liver. However, albumin mRNA was not detected in fetal or new born mouse kidney. Transient expression assays using AFP- and albumin-CAT (chloramphenicol acetyl transferase) vectors were employed to characterize cis-acting elements active in the regulation of AFP expression in kidney. Primary fetal liver and kidney cells in culture were used for these assays. The AFP promoter is active in kidney cells and the information necessary for tissue specific expression and developmental repression are contained within the first 1.0 kb of 5' flanking sequences of the AFP gene. In addition, the AFP upstream enhancer elements are inactive in primary kidney cells. The mouse albumin promoter is shown to be inactive in kidney cells. The results obtained using transient expression assays are consistent with the observed low level of AFP expression, developmental repression of AFP, and the absence of expression of albumin in the mouse kidney.


Nucleotide sequences of serine tRNAs from Bacillus subtilis.

Three B. subitilis serine tRNAs were sequenced including modified nucleosides. All the serine tRNAs contained 1-methyl-adenosine in the D-loop. As other characteristic modified nucleosides, 5-methoxyuridine was found in the first letter of the anticodon in the tRNA(UGA).


Mouse MRP8 and MRP14, two intracellular calcium-binding proteins associated with the development of the myeloid lineage.

MRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.


Inhibition of acute myelogenous leukemia blast proliferation by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors.

Interleukin-1 (IL-1) has recently been reported to play an important role in acute myelogenous leukemia (AML) blast proliferation. We therefore investigated the effect of soluble IL-1 receptors (sIL-1R) and IL-1 receptor antagonist (IL-1RA) on the growth of AML bone marrow blast progenitors from 25 patients. In the AML blast colony culture assay, sIL-1R and IL-1RA inhibited blast colony-forming cell replication in a dose-dependent fashion, at concentrations ranging from 10 to 500 ng/mL (sIL-1R) and 10 to 1,000 ng/mL (IL-1RA), and their inhibitory effect was partially reversed by IL-1 beta. A similar inhibitory effect was also noted with the use of anti-IL-1 beta neutralizing antibodies. When AML blast progenitors were grown either in the presence of fetal calf serum (FCS) alone or with one of the following: phytohemagglutinin leukocyte-conditioned medium (PHA-LCM), granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, interleukin-3 (IL-3), or stem cell factor (SCF), addition of 100 ng/mL sIL-1R or IL-1RA inhibited blast colony formation by 3% to 96% and 2% to 97%, respectively. In sharp contrast, neither of these IL-1-inhibitory molecules significantly inhibited proliferation of normal marrow hematopoietic progenitors. Lysates of 2 x 10(7) low-density AML marrow cells were tested for intrinsic IL-1 beta content using an enzyme-linked immunoadsorbant assay (ELISA). Samples from five of six patients showed high concentrations (ranging from 501 to 2,041 pg), whereas 2 x 10(7) cells from two normal marrow aspirates yielded 54.6 pg of IL-1 beta. AML blast colony-forming cells from all six patients were inhibited by sIL-1R, IL-1RA, or both. Incubation of nine samples of AML low-density cells with either sIL-1R or IL-1RA reduced GM-CSF concentrations in cell lysates, and supernatants from nine (P less than .01) and six samples (P less than .037), respectively, and G-CSF concentration in lysates from six of nine samples (P less than .03), and in supernatants from five of six samples (P less than .06) when studied by ELISAs. Our data implicate IL-1 in AML blast proliferation and suggest the potential benefits of using IL-1-inhibitory molecules in future therapies for AML.


Multilineage phenotypes of interleukin-3-dependent progenitor cells.

Interleukin-3 (IL-3)-dependent murine FDCP-mix cells have multilineage differentiation capacity; they are nonleukemic, have a normal karyotype, and are nonimmortalized. These cells coexpress on their cell surface the "early" B-lineage marker B220/CD45R and the myeloid marker Mac-1/iC3b receptor (CR3), transcribe germline T-cell receptor gamma genes, and express the macrophage lineage growth factor receptor gene c-fms as a predominant 8.4-kb transcript. They do not detectably express at the stable mRNA or protein level other lymphoid precursor cell genes including CD2, TdT, lambda 5, and BP1. Induction of granulocyte/macrophage differentiation in these cells closes down expression of the lymphoid genes and activates stable expression of genes specific to the myeloid lineage, including myeloperoxidase. Expression of the c-fms gene at the mRNA level is upregulated and the dominant stable transcript is now in the 4.1-kb form typical of the macrophage lineage. These data provide a plausible explanation for the coexpression of lymphoid and myeloid lineage markers on human leukemic cells of stem cell or progenitor cell origin and have implications for the programming of lineage potential in normal multipotential hematopoiteic progenitor cells.


Effect of inflammatory cytokines on hypoxia-induced erythropoietin production.

The effects of the inflammatory cytokines interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), and tumor necrosis factor-alpha (TNF-alpha) on erythropoietin (Epo) production in Hep3B cells were examined. The addition of IL-1 alpha, IL-1 beta, or TNF-alpha resulted in a dose-dependent inhibition of hypoxia-induced Epo production by as much as 89%. IL-1 beta was the most effective cytokine tested, demonstrating half-maximal inhibition at 0.4 U/mL compared with 1.0 and 10.0 U/mL for IL-1 alpha and TNF-alpha, respectively. TGF-beta also inhibited hypoxia-induced Epo production, but only by as much as 56%. In contrast to IL-1 alpha, IL-1 beta, TNF-alpha, and TGF-beta, the addition of IL-6 to hypoxic Hep3B cells resulted in a dose-dependent stimulation of hypoxia-induced Epo production by as much as 81%. However, IL-6 did not stimulate Epo synthesis in the absence of hypoxia, and was thus synergistic with hypoxia in inducing Epo production. Combinations of IL-1 alpha, TNF-alpha, and IL-6 were found to be additive in their effects on hypoxia-induced Epo production. By Northern blot analysis, Epo messenger RNA levels in Hep3B cells grown in 1% O2 were decreased when concurrently exposed to either IL-1 alpha or TNF-alpha. The effects that IL-1 alpha, IL-1 beta, TGF-beta, TNF-alpha, and IL-6 have on hypoxia-induced Epo production may provide new insights into the signal transduction pathway by which hypoxia leads to changes in gene expression. In addition, the effects of these inflammatory cytokines on hypoxia-induced Epo production in vitro suggest that in various inflammatory disorders these cytokines may affect Epo production in vivo and may play a significant role in the pathogenesis of the anemia of chronic disease.


Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets.

von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574----Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non-ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.


Maximizing opacification during excretory urography: effect of low-osmolarity contrast media.

Because of improved patient tolerance and decreased risks of idiosyncratic reaction, low-osmolarity contrast media are increasingly used for excretory urography. However, physiologic differences among patients may affect the optimal time for acquiring diagnostic radiographs during the pyelographic phase of the examination. A prospective, randomized, physician-blinded comparative study of 60 adult patients was undertaken to determine if the time to peak opacification of the pyelocaliceal systems differs with different doses of nonionic and ionic contrast media. Three doses of contrast media were used: a high dose (based on body weight) of a high-osmolarity ionic contrast medium, a high dose (based on body weight) of a low-osmolarity non-ionic contrast medium and a lower, fixed dose of a low-osmolarity nonionic contrast medium. The diagnostic quality of the radiographs did not differ statistically with the dose or the contrast medium. However, acquiring an additional radiograph during abdominal compression greatly increased the chance of obtaining at least one radiograph with maximal diagnostic information during the pyelographic phase. Despite potential differences among contrast media in the degree of pyelocaliceal opacification or distension and diuresis, it is not necessary to modify the timing of film acquisitions during excretory urography when lower doses of low-osmolarity agents are administered.


Is cerebral arteritis the cause of the Landau-Kleffner syndrome? Four cases in childhood with angiographic study.

Four children with Landau-Kleffner syndrome were studied over a six year period. They presented with acquired aphasia, epilepsy, and focal or generalized EEG discharges which were exacerbated during sleep. In addition, cerebral angiography demonstrated isolated arteritis of some branches of the carotid arteries in all cases. Computed tomographic and magnetic resonance images were normal. Nicardipine in a dose of 1 to 2 mg/kg/day, added to conventional anticonvulsant drugs provided effective supplementary control of seizures, of paroxysmal EEG discharges, and of language and behavioural disturbances, even several years after the onset of the disorder and in patients whose response to other medications, including steroids, had been poor. Interruption of nicardipine administration was followed by relapse of the language disorder. Repeat angiography was performed in all four patients and showed recanalization of obstructed vessels in two cases. Focal cerebral vasculitis may be the pathogenesis of the Landau-Kleffner syndrome and calcium channel blockers such as nicardipine may be effective and specific therapy.


Abnormal humoral immune response to mucosal antigenic stimulation in patients with lung cancer.

Previous studies have suggested an inverse relationship between atopy and cancer of mucosal surfaces. Atopy is classically assessed by detecting specific immunoglobulin E (IgE) antibodies against inhalant allergens. However, Platts-Mills recently proposed that atopy is the ability of an organism to recognize and to respond to limited doses of allergens presented at the mucosal level by producing not only IgE, but also immunoglobulin A and immunoglobulin G (IgG) antibodies. The authors compared the prevalence of atopy in 103 patients with lung cancer (a model of mucosal cancer), 51 patients with chronic obstructive pulmonary disease matched for age, sex, and smoking habits with patients with lung cancer, and 102 healthy control subjects. The authors investigated whether the IgG response to antigens presented at the mucosal level, exacerbated in atopic subjects, might inversely be decreased in patients with lung cancer. Serum IgE antibodies against five common inhalant allergens (Dermatophagoides pteronyssinus [Der p1], Aspergillus fumigatus, grass pollen, and cat and dog danders) were detected through a radioallergosorbent test assay. Serum IgG antibodies against allergens naturally presented at the mucosal level (respiratory mucosa with Der p1 and digestive mucosa with betalactoglobulin [BLG] and soya proteins [SP]) were measured through a solid phase enzyme-linked immunosorbent assay test. Atopic status was assessed in 19 patients (18.4%) with lung cancer, 9 patients (17.6%) with chronic obstructive pulmonary disease (COPD), and 18 healthy control subjects (17.6%). Distributions of specific IgG levels were represented on frequency histograms after natural logarithmic transformation and showed reduced levels of anti-Der p1 and anti-BLG IgG in the cancer population compared with the control populations but similar levels of anti-SP IgG. Influence of sex, age, smoking habits, histologic type of cancer, and its extent could be excluded. The authors' results show no difference in the prevalence of atopy between the three groups. They document a selective, rather than general, defect in the immune response initiated at the mucosal level in patients with lung cancer, the most frequent mucosal cancer in man.


Do histocompatibility antigens influence the risk of head and neck carcinoma?

Associations were sought between specific histocompatibility antigens (HLA) of the human major histocompatibility complex and the incidence of head and neck squamous cell carcinoma (SqCC). Seventy sequential patients with SqCC and 217 control subjects from the same geographic region were typed for HLA-A, HLA-B, and HLA-DR loci. These results were compared. Multivariate statistical analysis using stepwise logistic regression revealed significant associations between the incidence of SqCC and HLA-B14, HLA-DR3, and HLA-DR4 as well as smoking and the sex-smoking interaction. The authors concluded that certain host factors, including genetic constitution, and behavioral characteristics (i.e., smoking) as well as tumor biology, can influence the development of SqCC. The mechanism(s) of these associations may involve as yet undefined relationships between HLA region genes and the immune response.


Altered glycosylation of alpha-fetoprotein in hepadnavirus-induced hepatocellular carcinoma of the woodchuck.

Altered glycosylation of alpha-fetoprotein (AFP) has been proposed as a marker of hepatocellular carcinoma (HCC) in humans. The lectin-binding properties of woodchuck AFP were investigated to determine if woodchuck hepatitis virus (WHV)-induced HCCs are also accompanied by changes in AFP glycosylation. Ninety-eight to 100% of the AFP from normal, WHV-free woodchucks with physiologic AFP elevations and from WHV-carrier woodchucks with HCC bound to concanavalin A, indicating that virtually all of the AFP was glycosylated. Three percent or less of the serum AFP of normal woodchucks bound to Lens culinaris agglutinin (LCA). In contrast, the AFP from woodchucks with HCC had an increased LCA-binding fraction (range, 8-77%). The increased LCA-binding AFP in WHV-induced HCC is analogous to that which frequently accompanies hepatitis B virus (HBV)-induced HCC in humans. This study corroborates the relationship of altered glycoconjugate synthesis to virus-induced malignant transformation, confirms the importance of AFP glycoforms as markers of HCC, and demonstrates that the WHV-infected woodchuck should be useful in investigating changes in AFP glycosylation during hepadnavirus hepatocarcinogenesis and HCC growth.


Serum pancreatic enzyme assays in acute abdomen: a comparative prospective study.

Serum amylase, pancreatic isoamylase, lipase, trypsinogen and elastase-1 were measured in 100 consecutive patients who were emergency admissions to a surgical department, and in 27 selected patients with proven acute pancreatitis who served as controls. The final diagnoses in the 100 patients of the study group were: acute pancreatitis in eight patients, other digestive diseases in 87, and urogenital tract diseases in five. In the control group, pancreas-specific enzymes were abnormally high in all patients and amylase in 26 out of 27. In the study group, all enzymes were markedly high in all eight patients with acute pancreatitis. In the remaining 92 patients, serum amylase was abnormally high in seven, and at least one pancreatic enzyme was elevated in 16. These elevations were generally mild. The diagnostic efficiency, i.e., the percentage of patients correctly classified, was 96% for pancreatic isoamylase and lipase, 93% for amylase, 91% for elastase-1, and 84% for trypsinogen. We conclude that serum lipase turbidimetric assay is the most suitable test for emergency diagnosis of acute pancreatitis, because it is highly sensitive and specific and simply and quickly performed.