Presynaptic terminal loss from alpha-motoneurones following the retrograde axonal transport of diphtheria toxin. Intercostal motoneurones intoxicated following intraneural injection of diphtheria toxin exhibited a progressive dilatation and fragmentation of Nissl body rough endoplasmic reticulum (rER), coupled with two different forms of presynaptic terminal response. Firstly, terminal dysjunction without prior degeneration, and secondly, Wallerian-type degeneration. Dysjunction was attributed to a toxin-related failure by the motoneurones to maintain postsynaptic site structure. Degeneration was considered to arise from toxicity in presynaptic neurones, either neighbouring motoneurones or local interneurones. Morphometry revealed that by 8 days, intoxicated motoneurones exhibited a 33% loss in terminal frequency, a 15% loss in residual presynaptic membrane, and a 43% loss in overall presynaptic input. The concomitant loss of synaptic sites was greater that the overall loss of presynaptic membrane, indicating a toxin-related deficiency of the maintenance of postsynaptic sites. Analyses of the relationship between changes in terminal numbers and the development of Nissl body abnormality in the postsynaptic motoneurone identified three groups of motoneurones: (i) those with normal presynaptic input and normal neuronal Nissl body rER; (ii) those showing a dramatic loss of presynaptic input and a marked dilatation and fragmentation of Nissl bodies; and (iii) neurones exhibiting a maintained or further loss of presynaptic input coupled with extreme dilatation and fragmentation of Nissl body rER with loss of Nissl body structure. These changes are discussed in context with the known molecular action of diphtheria toxin.


Light microscopic response of neuronal somata, dendrites and axons to post-mortem concussive head injury. Forty anesthetized rats were cooled below 3 degrees C by 30-min transcardial perfusion of chilled physiological saline before a concussive head injury. The animals were then perfusion-fixed with a buffered formaldehyde-glutaraldehyde solution. Another forty rats were fixed by 30-min transcardial perfusion of the same fixative before a similar concussive head injury. In brain sections of both groups of animals a new silver method stained, in a Golgi-like fashion, a number of neurons and long axonal segments scattered among unstained ones. The similarity between these findings and those obtained following in vivo concussive head injuries described in accompanying papers suggests that the formation of traumatically induced argyrophilic neuronal damage is independent of metabolic processes, i.e., it may be a primary morphopathological process.


Immunohistochemical analysis of spinal intradural xanthomatosis developed in a patient with phytosterolemia. Multiple intradural xanthomatous tumors developed in 48-year-old female with familial phytosterolemia. These tumors were restricted to the spinal denticulate ligaments. Histological and immunohistochemical findings were fundamentally similar to those of tendinous xanthomas. The major cellular component of these tumors were identified as of mono-histiocytic origin because they possessed myeloid histiocytic antigen (Mac 387), CD11c and lysozyme but not CD15. Sitosterols, campesterols and cholestanols were recovered from the extract of the tumors and the lesions were confirmed to be phytosterolemic xanthomas. Schwann cells stained with anti-S100 protein were confined to the perivascular small nerve bundles and did not show xanthomatous change. Although immunohistochemical preparation of epithelial membrane antigen and desmoplakin I+II revealed the presence of non-neoplastic meningothelial cells in the superficial portion of the tumors, they were too few to play a significant role in the development of these xanthomas.


Etiologic and pathogenetic study of mental retardation with multiple congenital anomalies. Etiology and pathogenesis of MCA/MR in 1,023 patients (618 male; 405 female) with mental retardation were studied. Of 1,023 patients, there were 563 cases (317 male; 246 female) with MCA (55%). Among the MCA patients, there were 303 (156 male; 147 female) whose primary etiology was clarified (53.8%). Among the 260 patients with MCA/MR of unknown etiology, there were 23 with recognizable syndromes of unknown etiology and 7 previously reported by us as possibly having a new malformation syndrome. We had 569 patients with mental retardation of unknown etiology including 263 (41.5%) who were involved with MCA.


GC/MS analysis of urine in 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. A patient with 3-hydroxy-3-methylglutaric aciduria was diagnosed using gas chromatography mass spectrometry. The patient had severe metabolic acidosis, hypoglycemia and hyperammonemia and excreted abnormal amounts of 3-methylglutaconic, 3-hydroxy-3-methylglutaric, 3-methylglutaric, 3-hydroxyisovaleric and glutaric acids in the urine. 3-Hydroxy-3-methylglutaric acid appeared as two peaks on the chromatogram after trimethylsilylation. One was a tri-trimethylsilyl and the other a di-trimethylsilyl derivative. 3-Methylglutaconic acid appeared as three peaks: cis-, trans- and cyclic cis-isomers. The structure of these derivatives was elucidated by deuterium-labeled trimethylsilyl derivatization. The di-trimethylsilyl derivative of 3-hydroxy-3-methylglutaric acid and the cyclic cis-isomer of 3-methylglutaconic acid do not appear to have been previously described. After treatment with leucine restriction milk, the excretion of leucine catabolites decreased but 3-methylglutaconic and 3-hydroxy-3-methylglutaric acids continued to be excreted at abnormally high levels. It is concluded that these two metabolites are necessary for the chemical diagnosis of HMG-CoA lyase deficiency. This patient is the first case of HMG-CoA lyase deficiency to be reported in Japan.


[Review of 36 trigono-cervico-prostatectomies, carried out for prostatic pathology]. Review of 36 trigone-cervix-prostatectomies, performed in our services. Studies made before and after the procedure. Indications for trigone-cervix-prostatectomies. Complications and results obtained. Trigone-cervix-prostatectomies as an alternative to prostate transurethral resection, in a highly selected group of patients, because of its simplicity and better results.


[Morphological study of rubeosis iridis induced in animal eyes]. Rubeosis iridis was produced experimentally in rhesus monkey eyes, by means of occlusion of the major retinal vessels of the retina and persistent ocular hypotony. Clinically, rubeosis iridis was recognized 5 days after the procedure. Histopathologically, these vessels developed anteriorly to the iris surface and endothelial fenestrations showed evidence of iris neovascularization. Endothelial cells of the vessels projected toward the internal lumen and showed immaturity. Newly formed vessels originated from the stromal vessels in the iris. This method is an effective experimental model for the induction of rubeosis iridis.


[Unique immunological properties of short forms of the interphotoreceptor retinoid-binding protein derived uveitogenic peptide]. Interphotoreceptor retinoid-binding protein (IRBP) induces experimental autoimmune uveoretinitis in a variety of animals. We have previously shown that sequence 1169-1191 of bovine IRBP has strong uveitogenicity and immunogenicity in Lewis rats. In this study, two completely distinct antigenic sites were detected within a short form of this peptide. One site is localized in sequence 1182-1191. The second site localizes within sequence 1183-1191 and becomes detectable only when tryptophan at 1182 is deleted. Lymphocytes sensitized against the first determinant recognized a longer peptide as well as whole IRBP. Lymphocytes sensitized against the second determinant recognized only two peptides 1184-1191 and 1183-1191. No cross reactivity was detected between these two determinants. Amino acid substitution of tryptophan with alanine or glutamic acid at 1182 in peptide 1182-1191 caused complete loss of uveitogenicity and immunogenicity, while substitution with phenylalanine did not change any immunological activities of the original peptide. The unique immunological properties of IRBP-derived peptides were discussed.


[The natural history of serous retinal pigment epithelial detachment]. The natural history of 25 eyes of 21 patients with serous retinal pigment epithelial detachment (PED) within the vascular arcade was retrospectively studied to clarify the risk factors for development of choroidal neovascular membranes (NVM) from PED (mean age 55.8 +/- 10.4 years; mean follow-up 18.8 +/- 10.3 months). During the follow-up period, 6 eyes (24%) developed NVM. The significant risk factors for NVM development at the first visit were: older patient age (greater than 60 years): larger PED size (greater than 1 disc diameter): presence of PED in the fovea. Eyes with sensory retinal detachment that did not decrease during the follow-up had a significant high risk for NVM development. Eyes with NVM development showed significantly worse visual prognosis as compared to those with no NVM development. Life-table analysis (Kaplan-Meier method) showed that the incidence of developing NVM was 8% at 3 months, 16% at 6 months, and 24% at 9 months. These results suggested that the size and location of PED and clinical course of sensory retinal detachment in addition to the patient age may be important determinants of the prognosis of PED.


Symbols, rationality, and justice: rationing health care. Proposals to ration health care in the United States meet a number of objections, symbolic and literal. Nonetheless, an acceptance of the idea of rationing is a necessary first step toward universal health insurance. It must be understood that universal health care requires an acceptance of rationing, and that such an acceptance must precede enactment of a program, if it is to be economically sound and politically feasible. Commentators have argued that reform of the health care system should come before any effort to ration. On the contrary, rationing and reform cannot be separated. The former is the key to the latter, just as rationing is the key to universal health insurance.


Molecular cytogenetic and clinical studies of 42 patients with marker chromosomes. The molecular cytogenetic characterization and clinical details of 20 patients with marker chromosomes are presented. These 20 patients, together with another 22 patients previously published, represent a cohort in which the chromosomal origin of the marker chromosomes was successfully determined in all but one case. Examination of the pooled data suggests that the satellited markers derived from chromosomes 14, 15 (when metacentric or submetacentric), those whose origin is either 13 or 21, and those small ring autosomal markers derived from both alphoid and satellite II or III pericentric heterochromatin of chromosomes 1, 9, 15, and 16 are all associated with a low risk of phenotypic abnormality. The markers identified as i(18p), ring chromosomes derived from various autosomes, and satellited markers derived from chromosome 22 are associated with a high risk of phenotypic abnormality. The phenotype of patients with acrocentric markers derived from chromosome 15 was equivocal, perhaps as a result of imprinting. Additional data are required to confirm these trends. The mild mental retardation and abnormal face of a patient with a small ring chromosome derived from chromosome 4 are described. Identification of patients with small rings originating from particular chromosomes may allow the recognition of new syndromes.


Intracellular band 3 immunostaining in type A intercalated cells of rabbit kidney. Intercalated cells (ICs) in the collecting duct and the connecting tubule (CNT) are involved in H+ secretion and HCO3- reabsorption. H+ secretion is mediated by an H(+)-adenosinetriphosphatase in the apical plasma membrane, whereas a band 3-like Cl(-)-HCO3- exchanger in the basolateral membrane is responsible for HCO3- reabsorption. Recent studies have reported that a band 3-like protein is also present in mitochondria in rabbit ICs. The purpose of this study was to establish the subcellular location of the band 3-like Cl(-)-HCO3- exchanger in rabbit ICs by electron microscopic immunocytochemistry using a monoclonal antibody, IVF12, against erythrocyte band 3 protein. Rabbit kidneys were preserved by in vivo perfusion with a paraformaldehyde-lysine-periodate solution and processed for immunocytochemistry using a horseradish peroxidase preembedding technique. Band 3 immunostaining was observed on the basolateral plasma membrane of ICs in the outer medullary collecting duct and type A cells in the cortical collecting duct (CCD) and CNT. In addition, distinct staining for band 3 was present in numerous small vesicles and in multivesicular bodies in type A ICs in the CCD and CNT. However, there was no evidence of band 3 immunostaining of mitochondria or of the apical plasma membrane in any cells of the collecting duct. These observations suggest that basolateral Cl(-)-HCO3- exchangers in type A ICs in the rabbit kidney are stored in intracellular vesicles and possibly degraded in the vascular-lysosomal system when these cells are in a resting state. The previously reported band 3 immunolabeling of mitochondria could not be confirmed.


Insulin-like growth factor I in initial renal hypertrophy in potassium-depleted rats. We investigated insulin-like growth factor I (IGF-I) in the kidney during the initial renal enlargement induced by dietary K depletion in rats. Kidney weight increase was significant after 3 days of K depletion and amounted to 29% after 7 days compared with pair-fed controls [839 +/- 34 vs. 648 +/- 17 mg (SE), P less than 0.01]. The kidney growth occurred despite almost complete arrest in body weight gain in K-depleted animals (8 +/- 3 vs. 34 +/- 4 g/7 days in controls, P less than 0.01). Whole kidney protein, RNA, and DNA estimations indicated that cellular hypertrophy during the first 4 days was followed by hyperplasia. Immunoassayable kidney IGF-I concentration increased by 106% (673 +/- 30 vs. 327 +/- 14 ng/g, P less than 0.01) in K-depleted animals 24 h after induction of K depletion, stayed elevated until day 4, and returned to control levels on day 7. After K depletion for 24 h, IGF-I immunostaining was markedly increased in the medullary parts of the collecting ducts from K-depleted animals, whereas kidney IGF-I gene expression (IGF-I mRNA) had decreased by 36%. The increase in total kidney IGF-I concentration and immunostainable IGF-I in collecting ducts in kidneys from K-depleted rats precedes the renal hypertrophy and thereby suggests a renotropic role for IGF-I. The increase in kidney IGF-I concentration is not associated with increased IGF-I mRNA levels, indicating that non-transcriptional mechanisms may be responsible for the renal IGF-I accumulation.


Nephrotoxic antiserum identifies a beta 1-integrin on rat glomerular epithelial cells. A postulated mechanism of immune glomerular injury is a direct interaction between antibody and glomerular epithelial cell (GEC) surface antigens. To explore this hypothesis, we examined the interaction of the noncomplement-fixing gamma 2-subclass of sheep anti-rat nephrotoxic serum (NTS), which causes immediate complement- and neutrophil-independent proteinuria in vivo, with rat GECs in culture. Reactivity of NTS with GEC surface antigens was determined by positive immunofluorescence of GEC plasma membranes and by the ability of NTS-coated tissue culture wells to provide an adhesive substrate for GECs. NTS immunoprecipitated two proteins (135 and 118 kDa) from surface-labeled GECs. Proteins of similar molecular mass were precipitated by a polyclonal rabbit antibody that identifies the beta 1-integrin chain of the mouse fibronectin receptor (anti-FnR). In addition, NTS identified similarly sized bands on Western blot analysis of cell membranes from isolated rat glomeruli. Similar reactivity was eluted from the glomeruli of proteinuric rats injected with NTS. NTS significantly inhibited GEC adhesion to laminin, types I and IV collagen, and fibronectin and prevented GEC spreading on types I and IV collagen. Anti-FnR similarly inhibited GEC adhesion. Cell viability was not affected. These results show that NTS recognizes a pair of GEC surface proteins that have the characteristics of an alpha- and beta 1-integrin and, at low concentrations, disrupt cell-matrix interactions.


Substance P modulates autonomic nerve activity in canine hearts. We examined the hypothesis that substance P (SP) acts as an "afferent neuromodulator" in the heart regulating the response of the cardiac autonomic nerves to reflexes originating in the heart. We employed the acute, isovolumic canine heart preparation in which the amplitude of the chamber pressure accurately reflects changes in contractility. The heart was decentralized except for one-half of the right vagus, which was left intact to permit afferent communication with the central nervous system, while the remaining one-half was tightly ligated so that the distal part could be used for efferent stimulation. SP was injected in doses of 2-10 micrograms ic. There were no significant inotropic responses to 2 and 5 micrograms SP, whereas 10 micrograms produced positive inotropy of 5-15%. When vagal tone was elevated with sustained vagal stimulation, the same doses of SP increased contractility by 12-28%. Similarly, during right stellate ganglion stimulation (SS), SP decreased contractility 8-22%. After the intact half of the right vagus was sectioned, SP modulation of vagal responses was unaffected, while modulation of atrial, but not ventricular, responses to SS was significantly attenuated. When tested on a series of cardiac-denervated dogs, SP had no effect on cardiac inotropy at any dose. However, when contractility was increased with isoproterenol infusion, SP caused a small decrease in ventricular contractility. These results suggest that SP acts as a modulator of cardiac autonomic neural tone. It is possible that the neuropeptide is released from intramyocardial afferent collateral fibers and inhibits the elevation in vagal or sympathetic nerve activity initiated by activation of cardiac primary afferent nerves.


Cyclosporin inhibits mitochondrial calcium efflux in isolated adult rat ventricular cardiomyocytes. Exchangeable intracellular Ca2+ as measured by 45Ca2+ uptake more than doubled when isolated adult rat ventricular cardiomyocytes were incubated 30 min with 8 microM cyclosporin; nevertheless the cells retained a normal rod-shaped morphology. High concentrations of ouabain caused a similar increase in 45Ca2+ uptake, but in this case the Ca2+ overload caused nearly all cells to hypercontract into a round disorganized form. The response to cyclosporin was concentration dependent with an apparent half-maximal effective concentration of 0.5 microM for enhancement of net 45Ca2+ accumulation. Verapamil (1 microM) could not inhibit this cyclosporin effect, but it was abolished by a 5-min preincubation with 12 microM crude ruthenium red. Cyclosporin also decreased the rate of 45Ca2+ efflux from prelabeled myocytes into Ca(2+)-containing and Ca(2+)-free media. These data are consistent with inhibition of mitochondrial 45Ca2+ efflux through the cyclosporin-sensitive mitochondrial inner membrane pore. It would appear that periodic transient increases in mitochondrial inner membrane permeability provide a pathway for mitochondrial Ca2+ extrusion under relatively normal conditions in isolated adult rat heart cells.


Effect of Ca2+ ionophores on membrane potential of pig coronary artery endothelial cells. Ca2+ ionophores (A23187 and ionomycin) were used to determine whether an increase in cytosolic Ca2+ plays a direct role in pig coronary endothelial cell hyperpolarization. Ionophores induced concentration-dependent hyperpolarizations that were not altered by the presence of N omega-nitro-L-argnine (L-NNA), and inhibitor of nitric oxide synthesis. d-Tubocurarine decreased by 65-89% the A23187- and substance P (SP)-generated hyperpolarization of endothelial cells. To study the role of endothelial cell hyperpolarization in the endothelium-dependent relaxation of precontracted coronary artery strips, A23187 and SP concentration-response curves were built up in the presence of d-tubocurarine and/or L-NNA. A decrease in the maximal response was observed only when both d-tubocurarine and L-NNA were present. Our direct in situ approach gives results in agreement with a gating of Ca(2+)-activated K+ channels during A23187- and SP-induced hyperpolarizations of endothelial cells. We suggest that these hyperpolarizations play a role in the endothelial cell-dependent relaxation induced by A23187 and SP in the pig coronary artery.


Effects of hypertonic saline and dextran 70 resuscitation on microvascular permeability after burn. Administration of hyperosmolar fluids to burn patients has been proposed as a means of decreasing resuscitative fluid volume and, subsequently, wound edema accumulation. To test this hypothesis, canine hindpaw lymph flow (QL) and lymph (CL) and plasma (CP) total protein concentrations were measured, and capillary filtration coefficient (Kf) was calculated before and for 6 h after a 5-s 100 degrees C footpaw scald. Scald was followed 30 min later by bolus infusion (4 ml/kg) of 7% saline or 6% Dextran 70 or the two in combination. Before scald, venous pressure was elevated until a minimal CL/CP was reached. The reflection coefficient (sigma d) was calculated by the formula: 1-minimal CL/CP. Scald increased QL, CL/CP, and Kf, but sigma d was decreased (P less than 0.05). Compared with burn alone, 7% saline exacerbated burn-induced increases in QL and Kf. In contrast, infusion of Dextran 70 did not exacerbate these changes in QL or Kf. Perhaps more importantly, Dextran 70 may have attenuated the increase in CL/CP and reduced edema formation in the burned hindpaw. However, the addition of 7% saline to the dextran eliminated these beneficial effects.


Cardiomyocyte proliferation in mice expressing alpha-cardiac myosin heavy chain-SV40 T-antigen transgenes. To determine the proliferative potential of adult ventricular cardiomyocytes, we have generated transgenic mice that express the SV40 large T-antigen oncogene in the heart. A fusion gene comprised of the rat alpha-cardiac myosin heavy chain promoter and the SV40 early region was used to target oncogene expression to the myocardium. Expression of SV40 large T-antigen was observed in both atrial and ventricular cardiomyocytes in adult transgenic animals. T-antigen expression was associated with hyperplasia in the targeted cells. Immunohistological analysis indicated that the proliferating cells continued to express sarcomeric myosin. Electron microscopic examination demonstrated that cardiomyocytes in various stages of the cell cycle retained ultrastructural characteristics typical of mitotic cardiac muscle cells in vivo. Cardiomyocytes isolated from transgenic tumors were able to proliferate in culture and retained a differentiated phenotype, as evidenced by spontaneous contractile activity. Preliminary studies indicate that these cells can undergo a limited number of passages while retaining this differentiated phenotype. These studies demonstrate that both ventricular and atrial cardiomyocytes from transgenic mice proliferate in response to targeted T-antigen expression.


Reduction of glutamine synthetase mRNA in hypertrophied skeletal muscle. Skeletal muscle glutamine synthetase (GS) expression is reduced by endurance exercise and is increased when normal innervation is interrupted. This investigation was undertaken to determine whether GS expression is downregulated by the increased contractile activity associated with functional overload. Plantaris muscles overloaded for 30 days by synergist ablation were 70% heavier than those in sham-operated and unoperated control muscles. GS mRNA levels from hypertrophied muscles, measured by Northern and dot-blot hybridization, were reduced to 30% of controls. Changes in total RNA concentration and the proportion of poly(A)+ RNA in the total RNA pool did not account for the decline in GS mRNA. Despite reduced levels of GS mRNA, GS enzyme activity (nmol.h-1.mg protein-1) was unchanged in the hypertrophied muscles (overload, 79 +/- 5; control, 82 +/- 4). To further examine the lack of relationship between GS mRNA and enzyme activity, the concentration of glutamine, a known posttranslational modifier of GS activity, was measured. Consistent with the observed enzyme activities, muscle glutamine was unchanged in hypertrophied muscle (overload, 6.2 +/- 0.3; control, 5.8 +/- 0.4 mumol/g tissue). These results suggest that translational or posttranslational regulation, other than through alterations in glutamine concentration. may play a role in maintaining GS enzyme levels in hypertrophied muscle. Moreover, the regulation of GS activity in muscle hypertrophy may differ from the regulation with endurance training, in which changes in enzyme activity parallel changes in mRNA.


Human antibody responses to epitopes on the Plasmodium falciparum gametocyte antigen PFS 48/45 and their relationship to infectivity of gametocyte carriers. Antibodies in human sera recognizing epitopes I, IIa, III, and IV on the Plasmodium falciparum gametocyte antigen Pfs 48/45 have been investigated by competitive enzyme-linked immunosorbent assay. More than one-third of the residents of three villages in Madang, Papua New Guinea responded to epitopes I, IIa and III, with little variation by village or with time. There was a bimodal distribution of positive sera by age, with the highest proportion of responders in the 5-9- and greater than 20-year-old age groups. The data suggest a lower prevalence of antibodies against epitopes IIa and III in P. falciparum gametocyte carriers than in non-carriers. Enhancement of binding of monoclonal antibodies to epitopes IIa and III was also observed more frequently with sera from gametocyte carriers. Sera from gametocyte carriers in Papua New Guinea and Thailand, whose infectivity to mosquitoes had been tested, were used to examine the relationship between recognition of particular epitopes and infectivity. There was a significant association between lack of infectivity of P. falciparum gametocyte carriers and recognition of epitope IIa on Pfs 48/45 by antibodies in their sera.


Cortical synaptogenesis in congenitally hydrocephalic HTX-rats using monoclonal anti-synaptic vesicle protein antibody. We attempted to develop a method for investigating impairment of synaptogenesis quantitatively involving measurement of the fluorescence intensity emitted by immunohistochemically stained rat brain using a monoclonal antibody (Mab 171B5) against a synaptic vesicle protein (SVP-38). We applied this method to congenitally hydrocephalic and non-hydrocephalic brains of HTX-rats, and compared the postnatal changes in the fluorescence intensity in the molecular layer of the cerebral cortex. In non-hydrocephalic HTX-rats, the fluorescence intensity remained nearly unchanged from the 1st to 7th postnatal day and then increased at an almost linear rate until the 21st postnatal day, when it reached 4.5 times the value on the 7th postnatal day. The increase thereafter was gradual until the 28th postnatal day. In hydrocephalic HTX-rats, the fluorescence intensity increased nearly in parallel with that in non-hydrocephalic HTX-rats up to the 21st postnatal day. On the 28th postnatal day, the fluorescence intensity showed a marked reduction (P less than 0.01). This finding indicates that despite the presence of continuous pressure due to progressive ventricular dilatation after birth, synaptogenesis in the cerebral cortex may be relatively resilient.


A monoclonal antibody (ST-1) directed to the native heparin chain. A mouse monoclonal antibody, ST-1, was raised against heparin complexed to Salmonella minnesota. Characterization of this antibody showed that it recognizes an epitope in the intact molecule of heparin that is present regardless of its source or anticoagulant activity. ST-1 is the first monoclonal antibody specific for the intact unmodified molecule of heparin to be described. 3H-labeled heparin in solution was immunoprecipitated by ST-1, and the formation of the 3H-labeled immunocomplex was selectively inhibited by unlabeled heparin. No cross-reactivity of ST-1 was observed with other glycosaminoglycans such as heparan sulfate, chondroitin sulfate, hyaluronic acid, dermatan sulfate, and keratan sulfate, or with polyanionic polymers such as dextran sulfate. Selective removal of the N-sulfate groups or N,O-desulfation of heparin strongly reduced the binding of ST-1. Inhibition of binding was also observed after carbodiimide reduction of the carboxyl groups of the uronic acid units of heparin. Competitive assays of ST-1 binding to heparin immobilized on poly-L-lysine-coated plates using oligosaccharides of different sizes that arose from HNO2 cleavage of heparin showed that the minimum fragment required for reactivity of ST-1 is a decasaccharide.


A novel serum assay for breast epithelial antigen using a fusion protein. Serum levels of breast epithelial antigens (BrE-Ags) are presently used in the follow-up of breast cancer patients. Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity. A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of the NP5 fusion protein, produced in Escherichia coli, that is composed of beta-galactosidase and polypeptide sequence obtained from a breast carcinoma cell line cDNA library, and anti-human milk fat globule monoclonal antibody Mc5. The fusion protein carries an altered epitope sequence (mimotope) that is similar, but not identical, to that found in the native antigen. This new competitive assay configuration has two essential features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for Mc5 and a competitive step that provides quantitation. Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.


Immunodetection after complete destaining of coomassie blue-stained proteins on immobilon-PVDF. A technique that simplifies the localization of an immunodetectable protein in relation to the other electrophoresed proteins is described. Proteins are transblotted onto a polyvinylidene difluoride (PVDF) membrane and visualized by staining with Coomassie brilliant blue R-250, and a photograph of the protein pattern is taken. The Coomassie blue-stained PVDF membrane is then completely destained using a 25% acetic acid/50% methanol solution that allows subsequent immunostaining on the same membrane. The technique uses common laboratory reagents, is rapid, and has been shown to be applicable for a variety of proteins using both monoclonal and polyclonal antibodies and a variety of transblots.


Using microparticle labeling and counting for attomole-level detection in heterogeneous immunoassay. A new heterogeneous "sandwich" immunoassay utilizing microparticles as labels to realize high sensitivity is described. In this method, antibody fixed on the microparticles reacts with antigen previously trapped on a microplate surface, which makes the antigen molecules visible and countable with an inverted optical microscope. The method is highly sensitive because the reacted single microparticle, therefore single antigen molecule, can be detected. The sensitivity depends both on the reaction efficiency of the immunoreaction and on nonspecific adsorption of the microparticles on the microplate surface. Therefore, the protocol for preparing microparticle having antibody on the surface and a microplate having capture antibody was investigated to realize high sensitivity. Carboxylated microparticles of 0.76 microns in diameter were conjugated with affinity-purified antibody using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. It was determined that 1 g microparticles had 880 micrograms antibody (approximately 1100 antibody molecules per 1 microparticle). The immunoreaction efficiency reached 18% at 1 x 10(-13) mol/liter antigen concentration. The lower detection limit was 3.1 x 10(-14) mol/liter (1.6 amol) using human alpha-fetoprotein as a model antigen.


Cetyltrimethylammonium bromide discontinuous gel electrophoresis: Mr-based separation of proteins with retention of enzymatic activity. A discontinuous polyacrylamide and agarose gel electrophoresis system is presented here which allows the fine separation of proteins based on molecular weight with the concomitant retention of native enzymatic activity. This system, referred to as the CAT gel, uses the cationic detergent cetyltrimethylammonium bromide (CTAB) and includes a stacking gel based on the zwitterion arginine and the buffer N-tris(hydroxymethyl)-methylglycine. The CAT gel system allows specific enzyme histochemical detection and localization of proteins after gel electrophoresis. We present evidence that the CAT system stacked and separated a broad range of proteins into discrete bands which migrate as a linear function of log Mr. We have also assessed the effect of CTAB solubilization on the activity of several proteins and showed that some proteins separated by CAT electrophoresis maintain high levels of native enzymatic activity and may be detected histochemically in situ.


High-yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride-based membrane. In this report we describe the use of a novel, experimental, polyvinylidene fluoride-based membrane with a cationic surface for the isolation by electroblotting of small amounts of proteins separated by gel electrophoresis for further characterization by protein fragmentation for internal sequence analysis. The membrane is characterized by a surface that mediates primarily ionic protein/membrane interactions and that allows the recovery of adsorbed proteins at high yields under relatively mild conditions. In electroblotting experiments, the novel membrane has a binding capacity that is at least equivalent to that of standard polyvinylidene fluoride membranes and is compatible with both chemical and enzymatic fragmentation of blotted proteins in situ. Intact electroblotted proteins, or fragments thereof, were eluted at high yields. Further structural analysis is demonstrated using reverse-phase high-performance liquid chromatography or gel electrophoresis to separate cleavage fragments for either pulsed-liquid- or solid-phase automated sequence analysis.


Characterization of insulin-like growth factor binding proteins by two-dimensional polyacrylamide gel electrophoresis and ligand blot analysis. Insulin-like growth factor binding proteins (IGFBPs) in pregnant baboon serum and tissue culture media obtained following explant culture of uteri from pregnant baboons were characterized by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) followed by Western ligand blot analysis using 125I-labeled IGF-I. IGFBP-1 (Mr 30,000; pI 4-4.2), IGFBP-2 (Mr 34,000, pI 5.7-6.2), IGFBP-3 (doublet Mr 42-48,000; pI 6.2-6.8), and IGFBP-4 (Mr 24,000; pI 5.7-6.0) were clearly separated from one another. The authenticity of IGFBP-1, -2, and -3 was verified by immunoprecipitation using polyclonal antibodies followed by ligand blotting. Specificity of 125I-labeled IGF-I binding to IGFBPs was also determined by competitive binding studies using unlabeled IGF-I and -II. This technique allows for the identification of IGFBPs in complex biological fluids on the basis of their characteristic Mr and pI with or without the availability of specific antibodies and can be done rapidly using the mini 2D SDS-PAGE systems.


Large-scale purification of calf pancreatic zymogen granule membranes. A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.


Enzymatic synthesis of p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside as a substrate for human alpha-amylases. Enzymatic modification at the nonreducing end D-glucosyl residue of p-nitrophenyl alpha-maltopentaoside was developed by using the transglycosylation of beta-D-galactosidase from Bacillus circulans. The enzyme regioselectively synthesized p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside (a yield of 12.0% based on the amount of p-nitrophenyl alpha-maltopentaoside added) on a preparative scale from lactose as a donor and p-nitrophenyl alpha-maltopentaoside as an acceptor. It revealed that the nonreducing end galactosyl group of p-nitrophenyl 4(5)-O-beta-D-galactosyl-alpha-maltopentaoside did not prohibit the action of human salivary and pancreatic alpha-amylases. This derivative was shown to be very suitable as a novel substrate for analytical use of human alpha-amylase assay in serum through a conjugated reaction involving glucoamylase and alpha-D-glucosidase.


Critical threshold for tissue viability as determined by laser Doppler flowmetry. As a noninvasive method for continuous evaluation of tissue perfusion, laser Doppler flowmetry fulfills many of the prerequisites of an ideal objective monitor. A critical threshold for insuring flap viability, however, has not been universally accepted. In this experimental design using pedicled epigastric flaps in rats, it was determined that if flow alterations in an otherwise healthy flap always exceeded 30% of the initial baseline value, consistent viability could be predicted. Although this relative critical index could be established, the variability of even intraspecies flow rates precluded defining any specific numerical index of flow that could always predict flap survival.


C-erbB-2 expression in squamous cell carcinoma of the head and neck. Seventy-five squamous cell carcinomas of the head and neck were analysed for c-erbB-2 expression using immunohistochemical techniques with four different c-erbB-2 antibodies. No membrane staining was seen in any of the squamous cell carcinomas studied with any of the antibodies; however, c-erbB-2 cytoplasmic staining was seen in 60 per cent of the tumours. The significance of cytoplasmic staining is discussed and that it may possibly represent elevated c-erbB-2 expression in squamous cell carcinomas. C-erbB-2 cytoplasmic staining was also observed in 10 of 23 normal specimens obtained from the resection margin of the tumours. No correlations were found between positive c-erbB-2 cytoplasmic staining and any of the clinicopathological parameters or survival.


In vitro toxicological evaluation of ISIS 1082, a phosphorothioate oligonucleotide inhibitor of herpes simplex virus. ISIS 1082, a phosphorothioate oligonucleotide targeted to a translation initiation codon of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) virion capsid protein UL13 inhibits in vitro viral replication. To better understand the pharmacological properties of ISIS 1082, we examined its effects in nonvirally infected HeLa cells by using a number of cytotoxicity assays. Our data indicate that ISIS 1082 had no effect on HeLa cell viability as measured by cellular proliferation and clonogenic assays at concentrations as high as 100 microM. Additionally, DNA, RNA, and protein synthesis were only inhibited by 25% in cells treated with 100 microM ISIS 1082. The effects of ISIS 1082 on DNA synthesis were compared with those of acyclovir and trifluorothymidine, two clinically used antiherpetic agents. Acyclovir displayed effects similar to that of ISIS 1082. However, trifluorothymidine, which has been reported to be a potential mutagen and teratogen, significantly altered DNA replication at concentrations from 1 to 100 microM. Isolated HeLa DNA polymerases were inhibited by the compound, with a 50% inhibitory concentration of 2 microM. The in vitro antiviral (K. Draper and V. Brown-Driver, submitted for publication; K.G. Draper and V. Brown-Driver, Antiviral Res. Suppl. 1:106, 1991) and cytotoxicity studies suggest that ISIS 1082 is a selective, nontoxic, antiherpetic therapeutic agent.


Human breast carcinoma cell lines: ultrastructural, genotypic, and immunocytochemical characterization. Two new breast carcinoma cell lines, designated as UISO-BCA-1 and UISO-BCA-2, have been established from pleural effusions of postmenopausal women. Both cell lines show properties of mammary epithelial cells, such as positive immunoreactivity to cytokeratins and human milk fat globulin, presence of desmosomal junctions, numerous microvilli, intracytoplasmic duct-like vacuoles and tonofilaments. UISO-BCA-1 and UISO-BCA-2 cells differ from each other with respect to cellular morphology, ultramicroscopic details, immunoreactivity to Her-neu oncogene protein, chromosomal mode and in vivo and in vitro growth rates. UISO-BCA-1 cells are well-differentiated (as evident from their morphology and ultrastructural details) and hyperploid (42-114 chromosomes). In vitro, UISO-BCA-1 cells are fast growing, with a population doubling time of 31.2 +/- 9.6 hrs (n = 4), and are tumorigenic (100%) in athymic nude mice. In contrast, UISO-BCA-2 cells are poorly differentiated, but are also hyperploid, with 54-64 chromosomes. UISO-BCA-2 cells are slow growing in vitro (population doubling time: 56.0 +/- 5.0 hrs [n = 4]) and have limited tumorigenic potency (20-40%). Both these cell lines are estrogen and progesterone receptor (less than 10 fmol/mg protein) negative.


Mucoepidermoid carcinoma of the salivary glands: immunohistochemical distribution of intermediate filament proteins, involucrin and secretory proteins. Mucoepidermoid carcinomas of the salivary gland comprising low (n = 6), intermediate (n = 11) and high grade malignant (n = 11) tumors were evaluated for immunohistochemical reactivity of cytokeratin (monoclonal antibody KL1, PKK1, K8.12), vimentin, involucrin and secretory proteins (lysozyme, LY; lactoferrin, LA; alpha 1-antitrypsin, alpha 1-AT and alpha 1-antichymotrypsin, alpha 1-Ach). Keratin expression was usually confined to intense staining in epidermoid tumor cells, but was negative in almost all mucous cells. Vimentin was coexpressed with keratin only in epidermoid tumor cells. Great heterogeneity of intermediate filament proteins was found in intermediate and epidermoid tumor cells. Involucrin in epidermoid tumor cells was particularly abundant in well keratinized cells but was lacking in intermediate and mucous forming cells. The frequency of occurrence of positive staining for LY, LA, alpha 1-AT and alpha 1-Ach was relatively low in mucoepidermoid carcinoma with positive immunohistochemical staining for these secretory proteins in mucous forming tumor cells, while varying expression was observed in epidermoid tumor cells.


Identification and cloning of Bradyrhizobium japonicum genes expressed strain selectively in soil and rhizosphere. The growth of Bradyrhizobium japonicum USDA 110 and USDA 438 in soil extract-supplemented medium led to transcription of a large amount of DNA not expressed in basal medium. Strain USDA 438 was more competitive for the nodulation of soybean than strain USDA 110. To identify and isolate DNA regions which were expressed specifically in strain USDA 438 but not in strain USDA 110 in response to soil extract or soybean root exudate, we developed a subtractive RNA hybridization procedure. Several cosmid clones which showed strain-specific gene expression were isolated from a USDA 438 gene library. Two clones enhanced competitive nodulation when mobilized to USDA 110. The method described may be useful for identifying genes expressed in response to environmental stimuli or genes expressed differently in related microbial strains.


Pseudomonas cepacia suppression of sunflower wilt fungus and role of antifungal compounds in controlling the disease. In a field experiment, Pseudomonas cepacia J82rif and J51rif increased sunflower emergence in the presence of the fungus Sclerotinia sclerotiorum. Pyrrolnitrin, aminopyrrolnitrin, and monochloroaminopyrrolnitrin were isolated from J82 and identified by using thin-layer chromatography, high-performance liquid chromatography, and electron impact-mass, UV, and infrared spectroscopy. In growth chamber experiments, two antibiosis-negative mutants were not different from the parent strain in protecting the seeds from the fungus.


Phylogenetic position of Rhizobium sp. strain Or 191, a symbiont of both Medicago sativa and Phaseolus vulgaris, based on partial sequences of the 16S rRNA and nifH genes. Phenotypic and DNA sequence comparisons are presented for eight Rhizobium isolates that were cultured from field-grown alfalfa (Medicago sativa L.) in Oregon. These isolates were previously shown to nodulate both alfalfa and common bean (Phaseolus vulgaris (L.) Savi.). The objective of the present study was to determine their phylogenetic relationships to the normal symbionts of these plants, Rhizobium meliloti and Rhizobium leguminosarum biovar phaseoli, respectively. Phenotypically, the Oregon isolates more nearly resemble strains from P. vulgaris than those from M. sativa. For example, even though nitrogen fixation levels were low with both host species, the symbiotic efficiency of a representative Rhizobium isolate (Or 191) with common bean was twice that observed with alfalfa. Comparative sequencing of a 260-bp segment of the 16S rRNA gene (directly sequenced after amplification by the polymerase chain reaction) demonstrated that Or 191 is not closely related to the type strain of R. meliloti (ATCC 9930), R. leguminosarum (ATCC 10004), or Rhizobium tropici (CIAT 899). Instead, sequence comparisons of the 16S gene indicated that Or 191 belongs to a distinct and previously unrecognized taxonomic group that includes strains that have previously been called R. leguminosarum bv. phaseoli type I. Unlike type I strains, however, Or 191 has only a single copy of the nifH gene (type I strains have three), and the nucleotide sequence of this gene is substantially different from those of other rhizobial and nonrhizobial nifH genes examined thus far.


Proteinolytic activity against IGF-binding proteins involved in the paracrine interactions between prostate adenocarcinoma cells and osteoblasts. PA-III rat prostate adenocarcinoma cells are capable of inducing osteoblastic reaction after inoculation onto rat skeleton. In this study PA-III cells and osteoblast-derived rat osteosarcoma cells (UMR 106 cells) were employed to characterize the cellular interactions in the PA-III cell-induced bone tumors, in vitro. Insulin-like growth factor-I (IGF-I) and conditioned media (CM) of UMR 106 cells stimlated tritiated-thymidine incorporation into the DNA of PA-III cells growing in serum-free media. This effect was inhibited by monoclonal anti-hIGF-I antibody. In addition PA-III cell CM contained proteinolytic activity for the IGF-binding proteins of UMR 106 cell CM (IGFBP-1 and -2). This proteinase activity hydrolyzed also benzyloxycarbonyl-lysine thiobenzyl ester (BLT) and its action on IGFBPs and BLT was inhibited by benzamidine and aprotinin. Proteinase activity of PA-III cell CM when bound covalently to tritiated-dilsopropylfluoro-phosphate (DFP) and then analyzed on SDS-PAGE gel electrophoresis, revealed the presence of radioactivity linked with a 35 kDa protein band. This proteinase was eluted in the void volume of the G-50 sephadex column and was retained on and eluted from p-benzamidine affinity column. The 35 kDa proteinase was retained on and was eluted from cartridges of the C18 silica by 80% acetonitrile over 0.1% trifuroacetic acid. This partially purified material hydrolyzed BLT substrate and IGFBPs of UMR 106 cell CM and its effect was inhibited by benzamidine and aprotinin. These data indicate that PA-III cell CM contains a 35 kDa proteinase capable of digesting the IGFBPs and thus increases the bioavailability of osteoblast-derived IGFs. This mechanism may participate in the pathophysiology of the PA-III cell-induced bone tumor and its subsequent osteoblastic reaction.


Characterization of a methane-utilizing bacterium from a bacterial consortium that rapidly degrades trichloroethylene and chloroform. A mixed culture of bacteria grown in a bioreactor with methane as a carbon and energy source rapidly oxidized trichloroethylene and chloroform. The most abundant organism was a crescent-shaped bacterium that bound the fluorescent oligonucleotide signature probes that specifically hybridize to serine pathway methylotrophs. The 5S rRNA from this bacterium was found to be 93.5% homologous to the Methylosinus trichosporium OB3b 5S RNA sequence. A type II methanotrophic bacterium, isolated in pure culture from the bioreactor, synthesized soluble methane monooxygenase during growth in a copper-limited medium and was also capable of rapid trichloroethylene oxidation. The bacterium contained the gene that encodes the soluble methane monooxygenase B component on an AseI restriction fragment identical in size to a restriction fragment present in AseI digests of DNA from bacteria in the mixed culture. The sequence of the 16S rRNA from the pure culture was found to be 92 and 94% homologous to the 16S rRNAs of M. trichosporium OB3b and M. sporium, respectively. Both the pure and mixed cultures oxidized naphthalene to naphthol, indicating the presence of soluble methane monooxygenase. The mixed culture also synthesized soluble methane monooxygenase, as evidenced by the presence of proteins that cross-reacted with antibodies prepared against purified soluble methane monooxygenase components from M. trichosporium OB3b on Western blots (immunoblots). It was concluded that a type II methanotrophic bacterium phylogenetically related to Methylosinus species synthesizes soluble methane monooxygenase and is responsible for trichloroethylene oxidation in the bioreactor.


Identification of Brucella spp. by using the polymerase chain reaction. The application of two synthetic oligonucleotides as probes and as primers in the polymerase chain reaction is presented for a specific, sensitive, and quick identification of Brucella spp. The specific oligonucleotide sequences were chosen on the basis of a 16S rRNA sequence alignment between Brucella abortus and Agrobacterium tumefaciens.


The stimuli releasing histamine from murine bone marrow-derived mast cells (BMMC). 3. Effect of coculture with 3T3 fibroblasts on the histamine releasability of BMMC. Murine bone marrow-derived mast cells at 3 wk in culture were further cultured in the absence (N-BMMC) or presence (F-BMMC) of 3T3 fibroblasts in the medium containing LI-3, and were examined for their functional responses to either histamine releasing stimuli such as compound 48/80 or an inhibitor of histamine release, disodium cromoglycate. After 3 weeks in coculture with 3T3 fibroblasts, the mast cells increased their histamine content greater than 10 fold, and greater than 10% of the cells changed histochemically to become safranin positive. F-BMMC released approximately 10% histamine when challenged with compound 48/80 or substance P, whereas N-BMMC failed to do so. Furthermore, when the sensitized cells were challenged with DNP-HSA antigen, histamine release from F-BMMC but not from N-BMMC was inhibited by preincubation with disodium cromoglycate. We also examined changes in intracellular Ca2+ ([Ca2+]i) in the cells when challenged with compound 48/80. A transient increase in [Ca2+]i was observed on stimulation with the compound in F-BMMC but not in N-BMMC. Taken together, our results indicate that the interleukin 3-dependent cultured murine mast cells change functionally, as well as histochemically, into in vitro counterparts of connective tissue mast cells when cocultured with 3T3 fibroblasts and may be useful tools for analyzing the mechanisms involved in degranulation from connective tissue-type mast cells.


[Inhibitory effect of inhaled FK-506 on increased bronchial responsiveness and eosinophil infiltration in the airway mucosa]. We examined the effects of inhaled FK-506, a potent immunosuppressive agent, on increased bronchial responsiveness to acetylcholine and on eosinophil infiltration in a guinea pig models of asthma. The guinea pigs were sensitized by repeated inhalation of ovalbumin (OA). Twenty four hours after antigen challenge, bronchial responsiveness to acetylcholine significantly increased and a marked accumulation of eosinophils in the airways was observed. However, when the guinea pigs were treated with aerosolized FK (10 mg/ml) for 5 min per day for 6 successive days before antigen challenge, the increase in bronchial responsiveness was significantly suppressed and the eosinophil accumulation was strikingly reduced. Since inhaled FK significantly suppressed these responses, there is a possibility that inhaled FK may be a useful therapy for patients with chronic bronchial asthma in the future.


Stimulation of rat parotid secretion by cAMP analogues that synergistically activate the type II isoenzyme of the cAMP-dependent protein kinase. Beta-adrenergic-stimulated parotid secretion is believed to be mediated by activation of the cAMP-dependent protein kinase (PK-A). However, the relative roles of the type I and II PK-A isoenzymes are still unclear. Combinations of site-selective, lipophilic cAMP analogues that synergistically activate each PK-A were used to investigate this problem. The selectivity of synergistic activation with these combinations was verified with the partially purified parotid PK-A isoenzymes, using kemptide as a substrate. Synergism in activation of PK-AII was only seen with 8-thiomethyl cAMP (8-TM) and N-6-benzoyl cyclic AMP (N6B), while PK-AI was only synergistically activated by 8-(6-aminohexyl) amino cyclic AMP (AHA) and N6B. Additive activation of each isoenzyme was observed for the combination of 8-TM and AHA. Rates of amylase secretion from dispersed parotid acini in response to secretagogues were determined with a coupled enzyme assay for amylase activity, which was adapted for use in a microplate reader. Cells were stimulated to secrete during 30 min with different doses (0.1-1.0 mM) and combinations of the cyclic nucleotide analogues. Alone, N6B was most effective in stimulating amylase secretion. The basal amylase secretory rate was stimulated by these secretagogues (0.44 mM) to the following extent: 53-fold (N6B), 8-fold (8-AHA), 2-fold (8-TM). In combination at a series of concentrations, only 8-TM + N6B produced synergistic stimulation of secretion, while AHA + N6B and 8-TM + AHA did not.(ABSTRACT TRUNCATED AT 250 WORDS)


Limited accumulation of cyclic AMP underlies a modest vasoactive-intestinal-peptide-mediated increase in cytosolic [Ca2+] transients in GH3 pituitary cells. The 4-chlorophenylthio analogue of cyclic AMP evoked profound and long-lasting changes in cytosolic [Ca2+] ([Ca2+]i) in pituitary-derived GH3 cells. However, vasoactive intestinal peptide (VIP), a hormone considered to act via cyclic AMP, was ineffective in modulating [Ca2+]i. The ability of VIP to modulate [Ca2+]i was enhanced by treatments that increased intracellular cyclic AMP. Much greater concentrations of intracellular cyclic nucleotides were achieved by the analogue than with VIP, under any condition. Thus cyclic AMP may play a prominent role in regulating [Ca2+]i in these cells, but the ability of hormones to stimulate its synthesis is limited, leading to a weak action on [Ca2+]i.


Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, inhibits induction of acute-phase proteins by interleukin-6 alone or in combination with interleukin-1 in human hepatoma cell lines. Okadaic acid (OA), a specific inhibitor of protein phosphatases 1 and 2A, inhibited in a dose-dependent manner (5-20 nM) the induction of C-reactive protein (CRP), serum amyloid A (SAA) and fibrinogen by interleukin-6 (IL-6) plus interleukin-1 (IL-1), and of fibrinogen by IL-6 alone, in Hep 3B cells. Induction of alpha 1-proteinase inhibitor (alpha 1-PI) by IL-6 plus IL-1 or IL-6 alone was not significantly affected by OA up to concentrations of 20 nM, above which concentration OA was toxic in Hep 3B cells. OA also inhibited the induction of CRP, fibrinogen and alpha 1-PI by IL-6 in the NPLC/PRF/5 cell line, albeit at a higher concentration (80 nM). These results suggest that the signal transduction mechanisms regulating induction of acute-phase proteins by IL-6, either alone or in combination with IL-1, are mediated by activation of protein phosphatases 1 and/or 2A.


Growth hormone activates mitogen-activated protein kinase and S6 kinase and promotes intracellular tyrosine phosphorylation in 3T3-F442A preadipocytes. Physiological concentrations of growth hormone induced a rapid and transient activation of mitogen-activated protein kinase (MAP kinase) and S6 kinase in 3T3-F442A preadipocytes. These effects were abrogated by staurosporine and in cells chronically pretreated with phorbol esters, suggesting that protein kinase C is involved in the mechanism of activation. In addition, three cytosolic proteins exhibited a growth-hormone-dependent increase in tyrosine phosphorylation.


Glycogenolytic effect of pancreastatin in isolated rat hepatocytes is mediated by a cyclic-AMP-independent Ca(2+)-dependent mechanism. We have studied the effect of pig pancreastatin on glucose and lactate production in freshly isolated rat hepatocytes. Pancreastatin stimulated the rate of glucose output, whereas, in contrast with glucagon, it failed to modify the rate of lactate production. The effective concentration of pancreastatin was in the range 0.1-100 nM, with half-maximal rate close to 1 nM. The ability of pancreastatin to increase glucose output was abolished by chelation of the calcium in the medium. By itself, pancreastatin did not increase cyclic AMP (cAMP) levels and had no influence on cAMP levels in glucagon-stimulated hepatocytes. Our results point out a possible role of pancreastatin in glycogenolysis. This appears to be mediated by a cAMP-independent Ca(2+)-dependent mechanism.


Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa. In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.


Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors. We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.


cDNA cloning of human and rat brain myo-inositol monophosphatase. Expression and characterization of the human recombinant enzyme. Inositol monophosphatase (EC 3.1.3.25) is a key enzyme in the phosphoinositide cell-signalling system. Its role is to provide inositol required for the resynthesis of phosphatidylinositol and polyphosphoinositides. It is the probable pharmacological target for lithium action in brain. Using probes derived from the bovine inositol monophosphatase cDNA we have isolated cDNA clones encoding the human and rat brain enzymes. The enzyme is highly conserved in all three species (79% identical). The coding region of the human cDNA was inserted into a bacterial expression vector. The expressed recombinant enzyme was purified and its biochemical properties examined. The human enzyme is very similar to the bovine enzyme.


Epitope mapping reveals conserved regions of an auxin-binding protein. There is now good evidence that maize (Zea mays) auxin-binding protein (ABP) functions as a receptor. We have synthesized sequential overlapping hexapeptides to map the epitopes recognized by a number of antisera to ABP. Only a few regions of the protein are recognized, and these are shown to be exposed on the surface. Three epitopes predominate, and these are clustered around, but do not include, the glycosylation site. A comparison is made between these maps of sera against purified ABP, maps of sera raised against recombinant maize ABP expressed in Escherichia coli and computer antigenicity predictions. Our anti-(maize ABP) serum recognizes ABP counterparts in other plant species. We have used immunoblotting to affinity-purify the immunoglobulins which cross-react from the antiserum. Epitope mapping of these immunoglobulins suggests that two of the three predominant epitopes may be conserved in both monocotyledonous and dicotyledonous plants. The possible functional significance of these conserved epitopes is discussed.


Interaction of bleomycin with a bent DNA fragment. The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3' side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.


Cloning and expression of the thyrotropin-releasing hormone receptor from GH3 rat anterior pituitary cells. Functional thyrotropin-releasing hormone (TRH) receptors have been expressed in Xenopus laevis oocytes following the microinjection of total and poly(A)+ RNA from GH3 rat anterior pituitary tumour cells. Under voltage-clamp conditions, application of the peptide induced a biphasic Ca(2+)-dependent chloride current. The amplitude of the initial, fast, component of the response was dependent on the concentration of the hormone and on the amount of mRNA injected. Size fractionation of poly(A)+ RNA on a continuous sucrose gradient and Northern blot analysis indicated that the receptor was encoded by an mRNA of approx. 3.5 kb. A 3.28 kbp cDNA encoding the TRH receptor has been cloned and sequenced. Full functionality of the predicted 412-amino-acid receptor protein was demonstrated by functional expression of cell surface receptors in Xenopus oocytes after both cytoplasmic injection of sense RNA transcribed in vitro from this cDNA and nuclear injection of the cDNA under the control of the Herpes simplex virus thymidine kinase promoter. The predicted protein contains seven putative membrane-spanning domains and shows significant sequence identify with some G-protein-coupled receptors. RNA blot analysis indicates that the mRNA for the TRH receptor is exclusively expressed in the pituitary gland. Expression studies performed with clones in which the 3' region of the mRNA has been successively shortened indicate that the 3' terminal region is not an important determinant for efficient functional expression in oocytes.


Peptide-containing nerves in labial salivary glands in Sjögren's syndrome. The presence and spatial distribution of peptide-containing nerves in labial salivary glands from 10 Sjögren's syndrome patients were compared with those in salivary glands from 7 healthy controls. Immunoperoxidase staining was used to demonstrate vasoactive intestinal peptide (VIP)-immunoreactive (IR) fibers, postganglionic sympathetic fibers containing the C-flanking peptide of neuropeptide Y (CPON), and sensory fibers containing calcitonin gene-related peptide (CGRP) and substance P. Acini, intralobular ducts, small arteries, and postcapillary veins were richly innervated by VIP-IR fibers, whereas CPON-, CGRP-, and substance P-IR fibers were restricted to blood vessels. Peptide-containing nerves were found surrounding, but not in the middle of, the highly inflamed mononuclear cell areas. This topologic distribution suggests involvement of VIP-IR fibers in vascular, motor, and secretory components of the reflex salivary secretion, whereas the distribution and the vasoactive actions of CPON, CGRP, and substance P suggest a role in the regulation of the salivary gland circulation, and thus of transcapillary flow. Excessive release may contribute to a neurogenic inflammation. Local depletion and absence of trophic neuropeptide stimuli may contribute to acinar atrophy.


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Endothelial cell interactions with granulocytes: tethering and signaling molecules. The adhesion of granulocytes to endothelial cells requires regulated expression of molecules on both the endothelial cell and the granulocyte. These pro-adhesive molecules have diverse structures and mechanisms of expression, and act either to tether the two cells together or as signals that induce activation-dependent adhesion events. Combinations of tethering and signaling molecules regulate endothelial-cell-granulocyte interactions at the endothelial surface.


Distribution of DNA cleavages induced by bleomycin and neocarzinostatin in a defined sequence of rat glioma cells. We have demonstrated the usefulness of a highly reiterated sequence of rat DNA as a probe sequence for evaluating the effect of bleomycin (BLM) and neocarzinostatin (NCS) at the level of individual nucleotides. The 370 base pairs (bp) DNA fragment, purified from rat glioma C6 cells after Hind III digestion, was labeled with 32P at either the 3'- or the 5'-ends and then divided into 167 bp and 203 bp by Hae III. These end-labeled DNA fragments were reacted in vitro with BLM or NCS, and electrophoresed on the denaturating 8% polyacrylamide gels according to Maxam and Gilbert's sequencing protocol. BLM created DNA strand breaks at the guanine-cytosine and guanine-thymine (5'----3') sequences, and NCS cleaved DNA at the position of thymines and adenines. The highly reiterated sequence of rat brain tumor DNA therefore provides adequate knowledge of DNA damages induced by BLM and NCS.


Effects of vasoactive neuropeptides on human saphenous vein. To assess the role of neuropeptides in the control of vascular tone in the human saphenous vein the actions of substance P, vasoactive intestinal peptide, calcitonin gene related peptide, neuropeptide Y, and somatostatin on this blood vessel were examined. In vitro organ bath techniques were used with preparations of saphenous veins obtained from 29 patients (aged 41-66) who were undergoing coronary bypass surgery. Substance P, vasoactive intestinal peptide, and calcitonin gene related peptide relaxed pre-constricted vessels in a dose dependent manner with a rapid onset of action, taking one to two minutes to reach a plateau at each dose. Substance P (10(-9) to 10(-6) mol/l) induced relaxation with a maximum response (mean (SEM)) 23.0 (6.6)% of the total relaxation induced by glyceryl trinitrate 1 microgram/ml and a 50% maximal effective concentration of 6.8 x 10(-9) mol/l. Vasoactive intestinal peptide (10(-10) to 10(-7) mol/l) produced a relaxation of 27.0 (5.1)% at 10(-7) mol/l. The maximum responses induced by substance P and vasoactive intestinal peptide were significantly reduced, to 3.7 (2.8)% and 4.7 (2.0)% respectively, after removal of the endothelium. Calcitonin gene related peptide (10(-10) to 10(-7) mol/l) elicited only 14.3 (2.6)% relaxation at 10(-7) mol/l, and this was not affected by removal of the endothelium. By contrast, neuropeptide Y and somatostatin exerted concentration dependent constriction on resting vessels. Neuropeptide Y (10(-10) to 10(-7) mol/l) caused prolonged contraction (roughly 20 minutes to reach a maximum plateau at each dose). At 10(-7) mol/l, the constriction amounted to 28.0 (12.0)% of the response to 90 mM KCl, in ring segments with or without endothelium. Somatostatin (10(-10) to 10(-6) mol/l) quickly caused contraction with a maximum response of 42.7 (15.0)% and a maximum response of 42.7 (15.0)% and a 50% maximal effective concentration of 6.7 x 10(-6) mol/l. The constriction was greatly increased when endothelium was removed, with a maximum response of 78.2 (16.8)% and a 50% maximal effective concentration of 4.3 x 10(-7) mol/l. Vasoactive peptides have diverse effects on the vascular tone these effects are endothelium dependent. The exact physiological role and implication for performance of bypass grafts require further investigation.


Cardiac rupture after thrombolytic therapy: the use of aprotinin to reduce blood loss after surgical repair. Emergency cardiac surgery after recent thrombolytic therapy is associated with increased blood loss. A patient underwent emergency repair of a ruptured left ventricle after intravenous streptokinase treatment for acute coronary occlusion. High dose aprotinin was given during the operation to reduce the expected blood loss. Surgical repair was successful without bleeding complications. Total postoperative blood loss was 365 ml.


A comparative study on immunosuppressive effects of cyclosporin A and FK 506 on peripheral blood lymphocytes in dogs. Immunosuppressive effects of cyclosporin A (CsA) and FK 506 (FK) on peripheral blood lymphocytes were studied in dogs in respect to mixed lymphocyte reaction, proliferative responses to recombinant interleukin-2 (rIL-2), phytohemagglutinin (PHA) and concanavalin-A (Con-A); phenotypes of OKIa1, CD3, CD8 and surface IgM; cytotoxic activity against xenogeneic tumor cells. CsA (2.0 or 5.0 mg/kg, intravenously) or FK (0.16 mg/kg, intramuscularly) was given to mongrel dogs every morning for serial 21 days. The blood concentrations of CsA, measured as trough levels by fluorescence polarization method, ranged from 37 to 350 ng/ml in dogs administered at 2.0 mg/kg and from 170 to 894 ng/ml in dogs administered at 5.0 mg/kg during treatment, respectively. In dogs treated with FK at a dose of 0.16 mg/kg, the drug concentrations in the plasma during treatment ranged from 0.16 to 1.8 ng/ml. Mixed lymphocyte reaction and proliferative responses to rIL-2, PHA and Con-A, which were declined by CsA, were not affected by FK. In contrast, the proportion of OKIa1+ cells was not affected by CsA, whereas FK decreased the proportion of OKIa1+ cells progressively during the course of treatment. Cytotoxic activity was suppressed by both CsA and FK. These results possibly indicate that CsA and FK exert their immunosuppressive effects via different mechanisms.


Levels of human serum granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor under pathological conditions. Levels of serum granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with various leukocyte disorders were estimated by enzyme-linked immunosorbent assay (ELISA). Some cases of acute myelogenous leukemia and aplastic anemia showed elevated serum levels of G-CSF and/or GM-CSF, whereas almost all of 23 healthy controls showed G-CSF and GM-CSF levels lower than 100 pg/ml. High levels of both types of CSF were noted in patients with granulocytosis due to infection. These levels became lower after resolution of the infection. Daily changes in serum CSF levels were also examined in a patient with autoimmune neutropenia, and it was found that the peripheral neutrophilic granulocyte count changed almost in parallel with the serum G-CSF level but not with GM-CSF, following the pattern with a delay of about 4-5 h, suggesting the possibility that G-CSF mainly regulates peripheral neutrophil circulation.


Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy. The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF (KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumour growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228. These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.


Antigen-specific, MHC-unrestricted T cells. The published record suggests that in the majority of cases the antigen is recognized by the T cell receptor (TCR) as a complex of a foreign antigen and amino acid residues contributed by the major histocompatibility complex (MHC) antigens, and the antigen-specific, MHC-restricted effector function is an unambiguous result of this process. Alternatively, the T cell receptor may recognize a particular conformational form of the antigen which is dictated by the allelic differences in the MHC, resulting also in MHC-restricted recognition. When, however, a T cell which phenotypically fulfills all the requirements necessary to perform antigen specific, MHC-restricted function, shows a lack of MHC restriction, there are two possible explanations: 1) In addition to the MHC-restricted, antigen-specific T cell receptor the cell expresses, or has newly acquired the expression of another, MHC-unrestricted (NK-like) receptor, or 2) The specific antigen recognized by the T cell receptor, is able to bind to the receptor and activate the T cell without being presented by the MHC molecule. While the first possibility has been extensively described in the literature as well as other articles in this issue, the second possibility has not been dealt with to the same extent and is the primary focus of this review.


Characterization of the dengue virus-induced helper cytokine. Dengue type 2 virus (DV) induces a subpopulation of T lymphocytes of mouse spleen to secrete a soluble helper cytokine (HF) which enhances the DV-specific IgM antibody plaque forming cells (PFC). The present study undertaken to purify and characterize HF shows that it can be purified by low pressure liquid chromatography (LPLC) using Sephacryl S-200 column. HF consisted of two subunits, having a M(r) of 65-68 kDa on SDS-PAGE, and both had similar activity. The isoelectric point of HF was 6.5. HF-specific antisera (HFAS) raised in mice neutralized the activity of HF in mice, reacted with it in a Western blot assay, and bound HF in an immunosorbent column. HF bound to DV-antigen in an immunosorbent column and enhanced only the DV-specific PFC. HF had no effect on PFC against heterologous antigens such as Japanese encephalitis virus, Coxsackie B4 virus or sheep red blood cells. HF generated in mice of H-2k haplotype, enhanced DV-specific PFC in the same strain of mice but had no effect on that in the H-2d or H-2q haplotype strains of mice. Thus, DV-induced HF with a M(r) of 65-68 kDa, antigen-specificity and genetic-restriction differs from most of the similarly acting cytokines but appears similar to the cell-free form of T cell receptor alpha beta dimer.


Combined external beam and intracavitary radiotherapy in oesophageal carcinoma. Seventy patients (40 males, 30 female; age 36-87 years, median 72 years) who were unsuitable for surgery were referred to the Regional Radiotherapy Centre, Newcastle General Hospital with oesophageal carcinoma (54 squamous cell; tumour length 2-14 cm). Sixty-seven patients received combined external beam radiotherapy (20-50 Gy in 5-20 fractions over 1-4 weeks) followed by intracavitary radiation (66 patients received 10 Gy at 1 cm; the remaining 4 received less) using an endoscopically inserted afterloading technique at 6.4 Gy/hour. Swallowing was restored in 65 patients (92%), although 39 patients (55.7%) subsequently required dilatation. Four developed fistulae (1 day, 2 months, 11 months, 17 months post-treatment). The actuarial survival was 71% at 6 months and 42% at 1 year. The combined treatment is well tolerated, with few complications and provides good palliation.


The value of internal fixation and radiotherapy in the management of upper and lower limb bone metastases. Fifty-four consecutive patients underwent 61 orthopaedic operations for metastatic bone disease affecting the upper and lower limbs. These patients were subsequently managed using a consistent postoperative radiotherapy (RT) policy. There were 27 prophylactic internal fixations and 34 internal fixations of pathological fractures. There was a marked difference in survival between these groups. The median postoperative survival of the prophylactic (P) group was 15 months whereas that of the fracture (F) group was 2 months (P less than 0.0001). Ninety-three per cent of the P group and 59% of the F group were able to be discharged home following treatment. Subsequent local fracture requiring further surgical intervention occurred in 11% of the P group and in none of the F group. Seventy-eight per cent of the P group and 62% of the F group did not suffer any further sequelae at the operation site until the time of death or last follow-up. Patient mobility following surgery and RT for metastatic lesions occurring in the lower limb was significantly improved in both the P group (P less than 0.05) and in the F group (P less than 0.0001) such that 91% and 58%, respectively, of these patients were subsequently able to walk.


Frequent clonal abnormalities of chromosome band 13q14 in B-cell chronic lymphocytic leukemia: multiple clones, subclones, and nonclonal alterations in 82 midwestern patients. We performed cytogenetic analyses of peripheral blood lymphocytes from 82 Midwestern B-cell chronic lymphocytic leukemia (B-CLL) patients. The cells were cultured with mitogens for 3-4 days. At least 15 metaphase cells were analyzed in 79 (96%) cases. Fifty (63%) of the 79 patients had clonal chromosomal alterations. Structural modifications of the long arm of chromosome 13 at or near band 13q14 were the most frequent abnormalities, identified in 23 (46%) of the patients with clonal abnormalities. In several patients, the abnormality involving band 13q14 was the sole chromosomal alteration. There was a high incidence of complex karyotypes. Nine patients had multiple subclones that appeared to result from clonal evolution; seven patients had cytogenetically unrelated clones; three patients had both subclones and cytogenetically unrelated clones. Nonclonal abnormalities were also prominent. Our study confirms the high incidence of clonal abnormalities involving chromosome arm 13q and documents the clustering of abnormalities at band 13q14 in B-CLL. The evidence for clonal evolution and the presence of multiple unrelated clones in these patients suggest that B-CLL may not be a karyotypically stable disease.


Karyotypic evolution of a murine mammary adenocarcinoma in vitro and during progression from primary to metastatic growth in vivo. We have previously described a murine mammary tumor cell line (SP1) that metastasizes when transplanted into the mammary gland, but not when injected into the subcutaneous site. We used cytogenetic markers to assess genetic heterogeneity, and to monitor the selection and evolution of karyotypically distinct cell types during primary tumor growth and in metastases. The SP1 tumor cells are hypotetraploid (mean chromosome number = 72), and have at least four karyotypically distinct cell types. We found no consistent pattern of selection of tumor cell types in primary tumors. However, metastases were derived from a cell type that was present in the corresponding primary tumor. In addition, novel, karyotypically distinct cell types also appeared in the metastatic nodules. Markers that appeared in metastases included two translocations, t(10;18) and t(1;19). By injecting a mixture of cells from a metastatic nodule with a non-metastatic clone into mice, we showed that the new cell types in metastases displayed a stable increased growth and metastatic potential when compared to the non-metastatic clone, or when compared to the initial cell type from which the metastases derived. These results indicate that metastases are derived from a distinct cell type in the primary tumor, but that additional chromosome and cell evolution occurs, resulting in new cell types that are selected in metastases.


Linkage map of a region of human chromosome band 11q13 amplified in breast and squamous cell tumors. DNA amplification involving markers on human chromosome band 11q13 is a consistent feature of several major cancers, notably adenocarcinoma of the breast and squamous cell carcinoma of the head, neck, lung, and esophagus. Since the presence of the amplification may be clinically significant, by defining a subset of patients at increased risk, it is important to establish which of the several genes on the amplified DNA provides the selective force. Here we describe a physical map of the centromeric end of the amplified DNA as it exists in a particular squamous carcinoma cell line (UMSCC2) and establish an unambiguous order for several known markers in the region, including pMS51/D11S97, pHB159/D11S146, BCL1, PRAD1/D11S287, HSTF1/FGF4 and INT2/FGF3. Significantly, PRAD1 is within 120-150 kb of the BCL1 translocation breakpoint and the data identify a new CpG island (D11S814) between PRAD1 and HSTF1. The ordering of the HSTF1 and INT2 genes and the clustering of CpG islands in the region have important implications in assessing whether the frequently observed amplifications at 11q13 are centered on one or more genes.


dup(12)(q13----qter) in two t(14;18)-negative follicular B-non-Hodgkin's lymphomas. Two t(14;18)-negative follicular B-non-Hodgkin's lymphomas with the same chromosomal abnormality, dup(12)(q13----qter), are presented. The absence of a BCL2 gene rearrangement was confirmed by molecular studies in both cases. Instead, duplication of a 12q segment was found. Further evidence for the presence of the dup(12)(q13----qter) was found using fluorescence in situ hybridization. dup(12q) may be equivalent to the trisomy 12 originally described in B-chronic lymphocytic leukemia. This chromosome anomaly has also been reported in B-non-Hodgkin's lymphomas, usually in association with other chromosome anomalies and a more aggressive tumor phenotype. Occurrence of dup(12q) in two histologically similar cases of follicular small cleaved-cell lymphoma without a typical t(14;18), suggests that this karyotypic change may play a critical role in some cases of follicle center-cell lymphomas.


Malignant fibrous histiocytoma of the brain in a six-year-old girl. We have prepared karyotypes from a malignant fibrous histiocytoma (MFH) of the brain of a 6-year-old girl. Sporadic cases of MFH in the central nervous system have been reported. However, to our knowledge, this is the first central nervous system tumor to be subjected to cytogenetic analysis. The tumor demonstrated a complex karyotype, with a variety of numerical and structural abnormalities. Although no specific cytogenetic abnormality was observed, the karyotype of this case was similar to those reported for adult MFH of soft tissues.


Detection of amplified DNA sequences in human tumor cell lines by fluorescence in situ hybridization. An unambiguous and rapid characterization of amplified DNA sequences in tumor cells is important for the understanding of neoplastic progression. This study was conducted to evaluate the potential of fluorescence in situ hybridization (FISH) to identify such amplified DNA sequences in human tumor cell lines. Applying this technique, we followed the metaphase location and interphase position of amplified DNA sequences corresponding to the SAMK, MYC, and MYCN genes in four cell lines derived from human tumors: two gastric carcinoma lines (KATO III and SNU-16), a neuroblastoma (NUB-7), and a neuroepithelioma (NUB-20) line. In metaphase cells of KATO III, NUB-7, and NUB-20 lines, the amplified regions were clearly visible and easily identified at an intrachromosomal location: in KATO III and NUB-7 at a terminal position and in NUB-20 at an interstitial position. In SNU-16, on the other hand, the amplified SAMK and MYC sequences were identified in extrachromosomal double minute chromosomes (DMs). In this line, the SAMK and MYC sequences were coamplified in the same cells and were colocated on the same DMs. FISH also allowed the identification of amplified DNA sequences in nondividing cells, enabling us to distinguish, at interphase, whether the amplification gave rise to intrachromosomal amplified regions (IARs) or to extrachromosomal DMs. The FISH technique also allowed us to determine at metaphase as well as at interphase the extent of amplification and the size of the IARs.


Chromosome studies in plasma cell leukemia and multiple myeloma in transformation. Four patients with plasma cell leukemia (PCL) and two with multiple myeloma (MM) in transformation had complex numerical and structural chromosome abnormalities. From data published in the literature, the cytogenetic patterns of 46 cases of PCL or MM in the leukemic phase are compared with chromosomal abnormalities found in MM. Although the spectrum of chromosomal abnormalities is comparable in both diseases, the incidence of chromosome abnormalities is higher in PCL than in MM. Hypodiploidy with monosomies for chromosomes 13, 16, 17, and 18 is also more frequent in PCL than in MM. A mutation within the TP53 gene was detected in one of the three patients studied molecularly.


Rearrangements of chromosome arm 3q in poorly differentiated nasopharyngeal carcinoma. Cell lines were established from fresh tumor biopsies from two Saudi patients with poorly differentiated nasopharyngeal carcinoma (NPC). Cytogenetic analysis on Giemsa-banded metaphase cells revealed complex, abnormal karyotypes in both patients with modal chromosome numbers of 77 and 52. A der(3)dup(3)(q25-q2?7) or t(3;?)(q27;?) was observed in both cell lines. The rearrangements involving chromosomes X, 1, 4, 6, 7, 8, 12, 13, 15, 17, and 22 in the first patient and 1, 6, and 22 in the second patient could represent clonal evolution.


Hyperdiploidy arising from near-haploidy in childhood acute lymphoblastic leukemia. Acute lymphoblastic leukemia (ALL) of childhood is frequently characterized by a hyperdiploid karyotype. Typically, most of the affected chromosomes in the abnormal clone are present in three copies. We have studied two patients with hyperdiploid ALL whose leukemic cells were atypical in that all or most of the chromosomes were present in either two or four copies, raising a suspicion that the observed karyotype arose through duplication of chromosomes in a precursor cell with a near-haploid chromosome number. Analysis of restriction fragment length polymorphisms confirmed that both cases arose from a near-haploid cell; all informative disomic chromosomes tested had loss of heterozygosity. Furthermore, the hyperdiploid karyotypes did not arise via a perfect haploid cell with exactly 23 chromosomes, because tetrasomic chromosomes remained heterozygous. These two patients probably are classified best as near-haploid cases, which often are observed to have a co-existing hyperdiploid clone with a duplicated chromosome set. The distinction between typical hyperdiploidy and hyperdiploidy arising via a near-haploid cell may be clinically important, because the prognosis for patients with a hyperdiploid karyotype is favorable in comparison to that of patients with a near-haploid karyotype.


Loss of NF1 alleles in phaeochromocytomas from patients with type I neurofibromatosis. Type I neurofibromatosis (NF1) is a common autosomal dominant disorder that affects tissues derived from the neural crest. The manifestations are varied, comprising generalised disorders of growth and development as well as an increased risk of benign and malignant tumours including phaeochromocytomas and neurofibrosarcomas. The NF1 locus has been mapped to chromosome bands 17q11-12, and recently the NF1 gene has been cloned. Deletions identified in the constitutional genotype of some patients have suggested that the NF1 phenotype may arise from loss of function mutations of the NF1 gene, consistent with the hypothesis that it is a tumour suppressor gene. To date, however, analysis of NF1 tumours has not revealed the frequent allele losses encompassing the NF1 locus, implying loss of the wild-type NF1 allele, which would support this hypothesis. We report allele losses with markers flanking the NF1 region in each of 7 NF1 phaeochromocytomas. In each of the 3 tumours for which this could be determined, the loss involved the wild-type chromosome. These results provide strong evidence that, in cells of the adrenal medulla at least, the NFI gene may act as a tumour suppressor.


Molecular confirmation of BCR-ABL fusion in a chronic myeloid leukemia with a complex translocation involving chromosomes 9, 15, and 22. We performed molecular studies to resolve the status of BCR and ABL in the bone marrow cells of a CML patient with a Ph chromosome resulting from a complex translocation involving chromosomes 9, 15, and 22. DNA digestion with BamHI, HindIII, and BglII, followed by hybridization to a bcr-specific 32P-labeled probe, showed a rearranged banding pattern confirming the involvement of the bcr locus in the translocation. Furthermore, total cellular RNA isolated from the marrow was subjected to reverse transcription into cDNA and amplified by PCR with primers specific for BCR-ABL fusion cDNA. The amplified products obtained from this patient and from a CML patient with the standard t(9;22) were both of the expected length of approximately 317 bp.


Translocation t(5;19): a recurrent change in thyroid follicular adenoma. The cytogenetic study of a follicular thyroid adenoma revealed a t(5;19)(q13;q13.3). This is the second report of a translocation between chromosomes 5 and 19 in a thyroid follicular adenoma.


Conserved patterns of somatic mutation and secondary VH gene rearrangement create aberrant Ig-encoding genes in Epstein-Barr virus-transformed and normal human B lymphocytes. Expression of N-terminal-truncated Ig heavy chains without normal light chain expression has been shown to occur in human B cell tumor lines, and to be due to diverse types of structural alteration within the expressed Ig heavy and light chain genes. Due to the tumor cell origin of these lines, generation of aberrant Ig-encoding genes may only occur after malignant transformation, reflecting the release of the tumor B cell from the need to express functional Ig for continued clonal proliferation. The genetic basis for expression of VH-truncated mu chains without light chains in several Epstein-Barr virus (EBV)-transformed human B cell lines was investigated with the aim that this information would lead to detection of similarly aberrant Ig genes in normal human B lymphocytes. Analysis of the productive mu genes in three truncated mu-only human B cell lines showed a consistent structural change where a secondary VH-VHDJH gene rearrangement had occurred. The site of VH-VH joining was suggestive of a V(D)J recombinase-mediated event. A consistent pattern of mutation was also observed in the normal-sized, but non-functional kappa light chain transcripts, making them incapable of coding for a functional kappa chain. Using genomic DNA from peripheral blood lymphocytes of a donor whose B cells were originally used to make one of the truncated mu-only EBV B cell lines, a similarly mutated V kappa gene was detected and a similar composite VH-VH gene was cloned. The lack of such aberrant VH and V kappa genes in non-lymphoid cells of this individual showed that the structural abnormalities in the expressed Ig genes arose somatically during development of that B cell clone. The presence of these altered Ig heavy and light chain genes in the genomic DNA of untransformed lymphocytes also shows that generation of such variant B cell clones can be a pre-neoplastic event and occurs by genetic mechanisms active during normal B cell development.


The hinge region of the CD8 alpha chain: structure, antigenicity, and utility in expression of immunoglobulin superfamily domains. The lymphocyte surface CD8 antigen is a heterodimer with each chain containing a single Ig-related domain, a hinge-like sequence, a transmembrane segment, and a short cytoplasmic sequence. A soluble form of the rat CD8 alpha chain was produced by introducing a stop codon into the cDNA at the end of the region encoding the extracellular sequence and expressed in Chinese hamster ovary cells. sCD8 alpha was produced at 20 mg/l, and consisted of monomers, dimers, and higher aggregates. The latter could be minimized, but not eliminated, by removal of one of the two cysteine residues in the hinge region by mutation and by growth in serum-free medium. The positions of the N- and O-linked glycosylation sites and the disulphide bond in the Ig-like domain were determined. The MRC OX-8 antibody was shown to react with a region from the CD8 alpha hinge containing 24 amino acids and the antigenic determinant was sensitive to neuraminidase digestion. A construct encoding the Ig-like domain of rat CD8 alpha without the hinge was not expressed in CHO cells, indicating the importance of the hinge region for expression. It seemed possible that the CD8 alpha hinge might facilitate expression of other Ig-related domains and such expression could be detected using the MRC OX-8 antibody. To test the system cDNA constructs were made with the rat CD8 alpha hinge spliced to the V-like domain of mouse CD8 alpha, to the V alpha and V beta domains of a T lymphocyte antigen receptor, and to one or both of the Ig-like domains of the MRC OX-47 membrane antigen. All these forms were expressed as soluble proteins that were detected with the MRC OX-8 antibody. This method may prove useful for the expression of Ig superfamily domains for raising antibodies and other studies.