The immune response in viral encephalitis. The central nervous system (CNS) offers a unique organ system in which to study viral immunopathogenesis. The presence of the blood-brain barrier that restricts entry of cells and protein, the restricted expression of MHC antigens and the nonrenewable nature of the neuronal cell population offer challenges to the immune system for viral clearance and increase the chances for viral persistence. We have used Sindbis virus encephalitis in mice as a model system for the study of the development of immune reactions in the CNS and clearance of virus from neurons. The immune response to this and other viral infections of the CNS probably are initiated in peripheral lymphoid tissue followed by entry of activated T cells into the cerebrospinal fluid, meninges, and brain parenchyma. During Sindbis virus infection class I and II MHC antigens are expressed extensively on microglia which may present viral antigen produced by the infected neurons. Full development of the inflammatory response requires virus-specific T cells, but participating cells include NK cells, gamma delta T cells, monocytes and B cells. The entry of Ig-secreting B cells corresponds with the appearance of increased amounts of IgG and IgA in the cerebrospinal fluid. Clearance of Sindbis virus from the brain was studied using persistently infected severe combined immunodeficient (scid) mice. Passive transfer of immune serum or immune T cells to these infected mice demonstrated that antibody to a surface glycoprotein of the virus eliminated virus by a noncomplemented-mediated, noncytolytic mechanism. Immune T cells had no effect on virus replication.(ABSTRACT TRUNCATED AT 250 WORDS)


Principles of cytomegalovirus antigen presentation in vitro and in vivo. Cytomegaloviruses are members of the ubiquitous family of herpesviruses. They escape immunological clearance and persist throughout life in the infected host. Yet, the stability of the balance of this virus-host interaction is dependent upon the state of the cellular immune response, and usually requires the function of specific CD8 T lymphocytes. In a mouse model, the major antigen that triggers protective CD8 T lymphocytes has been characterized as a nonameric amino acid motif of a nonstructural virus protein. Analysis of the naturally presented peptide has led to the conclusion that the sequence of an antigenic epitope is not the only parameter that decides upon its efficient presentation. Furthermore, the virus has developed regulatory functions that can prevent antigen presentation in productively infected cells. Contradictions between in vivo and in vitro findings are resolved in a hypothesis postulating an essential contribution of cytokines to the in vivo control of productive cytomegalovirus infection.


[Conventional x-ray techniques and computed tomography in the diagnosis of mediastinal lymph node involvement in non-small cell bronchogenic cancer. Which method is reliable?]. 52 patients with bronchogenic carcinoma were studied preoperatively by means of conventional tomography and CT. The results were compared with surgical findings to evaluate the accuracy of these methods in detecting mediastinal lymph node involvement. 323 mediastinal lymph nodes were identified by CT and 237 of these were studied histologically. In 25% of lymph nodes with diameter between 0.5 and 1 cm metastases could be identified. On the other hand, 25% of lymph nodes measuring up to 3 cm did not contain metastases. The results varied in different regions (pre- and paratracheal, azygos region, aorto-pulmonary, subcarinal). The probability of involvement of small nodes was doubled if there were in regions neighbouring metastases. In view of the high incidence of false positive and false negative CT findings, conventional methods were sufficient in the presence of definite pathological findings.


[The place of computed tomography in the staging of bronchial carcinoma. A retrospective study]. The conventional radiological and CT findings in 347 patients with confirmed bronchial carcinomas have been compared. In 184 patients subjected to surgery, the post-operative TNM classification was compared with the pre-operative CT staging. It was found that CT staging of the T-classification was correct in 82% and of the lymph nodes in 62.5% of cases. CT had a sensitivity of 92% and 95% for the recognition of mediastinal and bronchopulmonary lymph nodes, respectively, and a specificity of 68% and 57%. In 163 patients not subjected to surgery the reasons for avoiding operation were also studied. In more than 80% of these patients it was the CT findings which had indicated that the patient was inoperable.


Viral inactivation in blood and red cell concentrates with benzoporphyrin derivative. Using both the vesicular stomatitis virus (VSV) and feline leukemia virus (FeLV) as models we have shown that the photosensitizer benzoporphyrin derivative ring A (BPD), when activated with red light (600-700 nm), is effective in eliminating both free virus and virally infected cells from spiked blood products and whole blood drawn from viremic cats experimentally infected with FeLV, under conditions which appear to share red blood cells. The effect of photodynamic therapy on infected lymphocytes, as visualized by scanning electron microscopy, initially appeared as a limited area of tiny holes in the membrane. These holes were subsequently seen to increase in size until the membrane appeared completely decomposed. The red cell membranes however, seem to be undamaged by such photodynamic treatment.


Selective formation of tumor necrosis factor-alpha (TNF) degradation products contributes to TNF mediated cytotoxicity. We compared tumor necrosis factor (TNF) metabolism by wild-type MCF-7 (WT) cells, by 40-fold doxorubicin resistant (40F) breast cancer cells and by PC3 and LNCaP prostate cancer cell lines. MCF-7 WT and LNCaP cell lines were sensitive to TNF cytotoxicity and both lines produced two major intracellular TNF degradation products of 15 kDa and 5.5 kDa. The MCF-7 40F and the PC3 cell lines were resistant to TNF and produced multiple TNF degradation products with molecular weights lower than 15 kDa. Both the breast and prostate lines showed TNF receptor crosslinking patterns consistent with a molecular weight of 55 kDa. The breast and LNCaP lines expressed TNF receptors with an apparent dissociation constant (Kd) of 0.4 to 0.6 nM, while the TNF resistant line had a Kd of 2 nM. Similar receptor numbers per cell were found for all cell types (4,000 to 8,000/cell), and comparable levels of TNF internalization were noted. TNF-conditioned medium from the TNF-sensitive cell types was cytotoxic toward both the TNF-sensitive and TNF-resistant lines, and the toxicity was significantly blocked by an anti-TNF monoclonal antibody. Hydrophobic interaction column HPLC fractionation of the TNF-degradation products produced by MCF-7 WT and LNCaP cells revealed that the trimeric, monomeric, and 5.5 kDa fractions possessed the greatest in vitro antitumor activity. These findings suggest that a TNF degradation product, produced selectively by TNF-sensitive cells, may contribute to the antitumor action of TNF.


Isolation and characterization of Rhizobium (IC3342) genes that determine leaf curl induction in pigeon pea. Nodulation by the Rhizobium strain IC3342 causes a leaf curl syndrome in certain tropical legumes such as pigeon pea (Cajanus cajan) (N.M. Upadhyaya, J.V.D.K. Kumar Rao, D.S. Letham, and P.J. Dart, Physiological and Molecular Plant Pathology 39:357-373, 1991). Transposon (Tn5) mutagenesis of this leaf curl-inducing (Curl+) Rhizobium strain yielded two Curl- Fix- and three Curl- Fix+ mutants. Plasmid visualization and subsequent Southern blot hybridization analyses with Tn5, nif and nod gene probes showed that the Tn5 element had inserted into the symbiotic (Sym) plasmid in three of the mutants. Restriction endonuclease analyses indicated that none of the Tn5 insertions were closely linked. Tn5-containing EcoRI fragments were cloned from each mutant and used as probes to isolate the corresponding wild-type DNA fragments from a cosmid (pLAFR3) genomic library. Fix+ and/or Curl+ phenotypes were restored in each mutant by the introduction of cosmids containing the corresponding wild-type DNA. A closely related but Curl- Rhizobium strain ANU240 was shown, by Southern hybridization, to contain conserved DNA sequences of all but one of the identified genetic regions of the Curl+ Rhizobium strain IC3342. Cosmids containing the genetic region unique to the strain IC3342, designated lcr1, conferred a Curl+ phenotype on the strain ANU240. DNA sequence analysis of the cloned lcr1 region revealed five open reading frames (ORFs). The ORF2 showed homology with the Escherichia coli regulatory gene ompR, and ORF4 showed homology with E. coli and Rhizobium meliloti regulatory genes fnr and fixK, respectively.


Analysis of the pelE promoter in Erwinia chrysanthemi EC16. The pelE gene of Erwinia chrysanthemi strain EC16 encodes an extracellular pectate lyase protein that is important in virulence on plants. Control of pelE expression is complex, because the gene is regulated by catabolite repression, substrate induction, and growth-phase inhibition. A Tn7-lux reporter gene system was employed to define DNA sequences comprising the pelE promoter. When EC16 cells were grown on medium containing sodium polypectate, pelE transcriptional start sites were observed only at 95 and 96 bases upstream of the translational start site. However, DNA sequences required for pelE expression were also shown by deletion analysis to reside between 196 and 215 base pairs upstream of the translational start site. In addition to these upstream elements, two putative operator sequences that interact with negative regulatory factors occurred downstream of the transcriptional start. Finally, deletion of three bases from a putative catabolite gene activator protein binding site in the pelE promoter eliminated activity. The data demonstrate that the pelE promoter is complex and suggest that it interacts with several regulatory proteins.


Receptors for platelet-derived growth factor on microvascular endothelial cells. Endothelial cells are widely thought to be unresponsive to platelet-derived growth factor (PDGF) and devoid of PDGF receptors. However, in examining the growth factor responses of microvascular endothelial cells isolated from human omental adipose tissue, we detected PDGF-induced tyrosine phosphorylation of an 180-kD glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of 125I-PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors/cell with a kD of 0.14 nM. PDGF stimulated tyrosine phosphorylation of PDGF and other cellular proteins in a dose- and time-dependent manner, with half-maximal receptor phosphorylation occurring at 0.3 nM recombinant human PDGF-BB within 1 min of PDGF exposure. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kD protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. The finding of functional PDGF receptors on human microvascular endothelial cells suggests an important direct role for PDGF in neovascularization.


Quinolinic acid: effects on brain catecholamine and c-AMP content during L-dopa and reserpine administration. The catecholamine content in rat brain tissue was determined following the administration of quinolinic acid alone or combined either with L-dopa and decarboxylase inhibitor or reserpine. Quinolinic acid alone decreased the levels of dopamine and noradrenaline, as well as those of c-AMP, and increased those of adrenaline. Treatment with L-dopa/decarboxylase inhibitor reversed the suppressing effect of quinolinic acid on dopamine, but not on noradrenaline. Reserpine alone depleted the contents of dopamine, noradrenaline and adrenaline. It could be concluded from the effects of quinolinic acid and reserpine given together that quinolinic acid suppresses the depletion of amines induced by reserpine. It has been demonstrated that quinolinic acid leads to injuries of nerve-cell bodies in pars compacta of the substantia nigra and in the striatum. Quinolinic acid is a natural metabolite of tryptophan, normally occurring in the liver, kidney and brain (Wolfensberger et al. 1983; Moroni et al. 1984). This compound exhibits convulsant and neuron excitant properties (Stone et al. 1987). It induces a selective pattern of neuronal degeneration both at the site of intracerebral injection (Schwarcz et al. 1983; Stone et al. 1987) and after general (intracardiac) administration (Beskid and Markiewicz 1988). The ability of quinolinic acid to produce neurotoxicity was greater in the striatum than in other parts of the brain. This prompted us to study catecholamine and c-AMP levels in rat brain tissue following quinolinic acid and L-dopa administration, as well as the influence of reserpine on quinolinic acid action.


Multiple sequence elements are involved in RNA 3' end formation in spleen necrosis virus. The function of the poly(A) signal in spleen necrosis virus (SNV) is dependent upon the distance between the cap site and the poly(A) site, while the function of the SV40 late poly(A) signal is independent of the distance. Deletions in the SNV poly(A) sequence do not alter the distance-dependent function. SNV/SV40 chimeric poly(A) signals show intermediate behavior between the SNV and SV40 poly(A) signals. These results indicate that multiple sequence elements are involved in the functions of either the SNV or SV40 poly(A) signals. This intermediate behavior is also observed with poly(A) signals from the mouse alpha-globin and herpes simplex virus thymidine kinase genes.


Asthma. Anti-inflammatory therapies.

Asthma. Anti-inflammatory therapies. It is likely that airway inflammation has an central role in the development and maintenance of the structural and functional abnormalities that characterise asthma. The recognition that asthma is a chronic inflammatory disorder of the airways has resulted in an increased emphasis on the use of anti-inflammatory drugs. The principal anti-inflammatory drugs are corticosteroids and to a lesser extent sodium cromoglycate and nedocromil sodium. Several other drugs and therapies may also have anti-inflammatory effects in asthma. The early use of prophylactic anti-inflammatory agents is advocated in the treatment of chronic asthma. It is hoped that this treatment scheme will result in a reduction in the morbidity and mortality from this common chronic condition.


Asthma. Airway smooth muscle.

Asthma. Airway smooth muscle. It has been recognised for some considerable time that contraction of airway smooth muscle is largely responsible for the bronchospasm that is a characteristic feature of the acute phase of an asthmatic attack. It is also well accepted that airway smooth muscle of asthmatic subjects is hyperresponsive to a wide range of provoking stimuli. One might anticipate, therefore, that a substantial amount would be known about the physiological role of airway smooth muscle. Surprisingly, this is not the case. The purpose of this article, therefore, is to provide an overview of airway smooth muscle with special reference to contractile mechanisms since they are, clearly, of relevance in asthma. In addressing this remit the following subject matter has been covered--structural aspects of the airway smooth muscle cell, electrical and calcium ion channel characteristics of the cell membrane, signal transduction mechanisms in relation to excitation-contraction coupling and the biochemical basis of contraction.


GABAergic synaptic current in dissociated nucleus basalis of Meynert neurons of the rat. gamma-Aminobutyric acid (GABA)-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were recorded from dissociated rat nucleus basalis of Meynert neurons which still had their synaptic boutons attached. The membrane currents were recorded by the whole-cell patch-clamp technique. Elevated extracellular K+ concentration and the addition of the calcium ionophore, A23187, enhanced the amplitude and frequency of spontaneous IPSCs. Ryanodine and Ca(2+)-free external solution containing EGTA or BAPTA markedly decreased the spontaneous IPSC activities. Spontaneous IPSC activities were reversibly reduced by baclofen and increased by phaclofen, indicating that the GABAB receptor regulates the release of GABA from nerve terminals and acts as a negative autoreceptor.


Differential influence of local anesthetic upon two models of experimentally induced peripheral mononeuropathy in the rat. Neuropathic pain remains a major complication of various forms of injury to peripheral nerves in humans. Recently, 2 models of peripheral mononeuropathy in the rat have been described which closely resemble the human condition. Bennett and Xie3 described one model induced by the placement of 4 loose ligatures around the entire sciatic nerve; Seltzer et al. have described a second model produced by the placement of a tight ligature around one-third to one-half of the sciatic nerve. It is the purpose of this work to compare the effect of these injuries on the time course and magnitude of hyperalgesia as measured by paw withdrawal latency to a radiant heat stimulus. In addition, to evaluate the hypothesis that neuropathic pain develops as a result of injury-associated discharges, some injuries were induced following anesthesia of the sciatic nerve. Our results show that the partial constriction neuropathy (PCN) described by Bennett and Xie3 develops in a faster time frame than that produced by the tight ligature, or partial transection neuropathy (PTN), described by Seltzer and co-workers. In addition, the PCN shows a reduction in both the duration and magnitude of behavioral hyperalgesia obtained for those animals in which local anesthetic (lidocaine) was applied to the sciatic nerve, while the PTN does not show this sensitivity. The data suggest that injury-related discharge is one important factor contributing to the generation of hyperalgesia in the PCN model. The mechanism(s) responsible for the generation of hyperalgesia in the early stages of the PTN model are not lidocaine-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)


Selective localization of C atrial natriuretic peptide receptors in the rat brain. We studied the distribution of C atrial natriuretic peptide (C-ANP) receptors in the rat brain after incubation with 125I-rANP, and competition with the selective C-ANP receptor competitor C-ANP(4-23) (10(-6) M). C-ANP receptors were selectively localized to a few structures, the external plexiform layer of the olfactory bulb, the arachnoid mater and the choroid plexus, and their amount corresponds to 25%, 75% and 35%, respectively, of the ANP specific binding. These data suggest specialized functions for C-ANP receptors in brain.


Equine arteritis virus: an overview.

Equine arteritis virus: an overview. The causative agent of the respiratory disease equine viral arteritis is a small, single-stranded RNA virus with a genome organization and replication strategy related to that of coronaviruses and toroviruses. Clinical signs of infection in horses vary widely and severe infection can lead to pregnant mares aborting. Infected horses generally make good recoveries but stallions may become semen shedders of equine arteritis virus (EAV). These carrier stallions play an important role in the dissemination and perpetuation of EAV. Laboratory tests exist to detect virus and the equine immune response to infection. However, vaccines are not currently licensed in the UK to combat viral arteritis, the incidence of which may increase due to changes in European legislation.


Autoradiographic analysis of [35S]TBPS binding in entorhinal cortex-kindled rat brains. A quantitative autoradiographic analysis of [35S]t-butylbicyclophosphorothionate (TBPS) binding to the gamma-aminobutyric acid (GABA)-mediated chloride ionophore was carried out in 104 brain areas of entorhinal cortex-kindled and control rats. Subjects were sacrificed either 24 h or 28 days after the last kindled seizure. Kindled subjects in the 24 h group showed reductions in mean [35S]TBPS binding in the lateral nucleus of the amygdala (-31%), the infralimbic cortex (-14%), and the paracentral nucleus of the thalamus (-22%). At 28 days, reductions in binding were observed in the infralimbic cortex (-15%) and the paracentral nucleus of the thalamus (-18%). These data suggest that repeated seizures (kindling) modify the GABA-mediated chloride ionophore, and that in some brain areas related to seizure generalization the modifications are very long lasting.


Glucose enhances recovery of potassium ion homeostasis and synaptic excitability after anoxia in hippocampal slices. Hippocampal slices exposed to brief anoxia combined with elevated glucose exhibit greater postanoxic recovery of synaptic transmission. Glucose may have improved recovery of synaptic transmission by enhancing the production of metabolic energy during and after anoxia. This enhancement should provide more ATP for energy-requiring ion transport processes, and lead (1) to a delayed onset of complete depolarization of CA1 pyramidal cells during anoxia (anoxic depolarization) and (2) to greater ion transport activity following anoxia. A delay in anoxic depolarization would protect neurons from damage if the duration of anoxic depolarization was shortened. Greater postanoxic ion transport would allow the re-establishment of ion gradients supportive of neuronal and synaptic excitability. The effects of glucose and anoxia on ion homeostasis and synaptic transmission were examined in rat hippocampal slices exposed to different glucose concentrations (5-20 mM). The duration of anoxic depolarization was held constant so that postanoxic damage related to this duration was controlled. We found that K+ transport and recovery of synaptic transmission after anoxia in hippocampal slices improved as glucose concentration increased. Also, anoxic depolarization was delayed as glucose concentration increased. Thus, added glucose may improve postanoxic recovery of synaptic transmission by better supporting ion transport.


Corticotropin releasing hormone related behavioral and neuroendocrine responses to stress in Lewis and Fischer rats. We have recently shown that susceptibility to streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats is related to a lack of glucocorticoid restraint of inflammation while the relative SCW arthritis resistance in histocompatible Fischer (F344/N) rats is related to their greater hypothalamic-pituitary-adrenal (HPA) axis response. The difference in pituitary-adrenal responsiveness results from decreased inflammatory mediator-induced hypothalamic corticotropin-releasing hormone (CRH) biosynthesis and secretion in LEW/N rats. Because CRH not only activates the pituitary-adrenal axis, but also is associated with behavioral responses that are adaptive during stressful situations, we wished to determine if the differential LEW/N and F344/N CRH responsiveness to inflammatory mediators could also be associated with differences in neuroendocrine and behavioral responses to physical and emotional stressors. In this study, LEW/N rats exhibited significant differences compared to F344/N rats, in plasma adrenocorticotropin hormone (ACTH) and corticosterone responses during exposure to an open field, swim stress, restraint or ether. Furthermore, hypothalamic paraventricular CRH mRNA expression was also significantly lower in LEW/N compared to F344/N rats after restraint. These differences in neuroendocrine responses were associated with differences in behavioral responses in LEW/N compared to F344/N rats in the open field. Outbred HSD rats, which have intermediate and overlapping arthritis susceptibility compared to LEW/N and F344/N rats, exhibited intermediate and overlapping plasma corticosterone and behavioral responses to stressful stimuli compared to the two inbred strains. These data suggest that the differences in CRH responses in these strains may contribute to the behavioral and neuroendocrine differences we have observed. Therefore these strains may provide a useful animal model for studying the relationship between behavior, neuroendocrine and inflammatory responses.


Recent advances in avian virology.

Recent advances in avian virology. Selected, recent research on the following avian diseases, and their causative viruses, has been reviewed: chicken anaemia, infectious bursal disease, turkey rhinotracheitis, avian nephritis, fowlpox, influenza, infectious bronchitis and turkey enteritis.


Alterations of plasma membrane fatty acid composition modify the kinetics of Na+ current in cultured rat diencephalic neurons. Properties of inward Na+ currents (INa) were examined in dissociated diencephalic neurons whose plasma membrane fatty acid composition had been altered. These neurons were grown in a defined medium supplemented with essential fatty acids (EFA) of either the w3 class (linolenic acid, 18:3w3) or the w6 class (linoleic acid, 18:2w6), which resulted in a two-fold increase in the plasma membrane phospholipid polyunsaturated fatty acid (PUFA) concentration. The properties of the inward INa of these neurons were compared with those of control neurons grown in the absence of any supplemented fatty acids. The INa of neurons supplemented with a non-essential fatty acid (NFA) of w9 class (oleic acid, 18:1w9) was also examined. Several properties have been modified to different degrees. The ratio of the amplitudes between the fast and the slow decay components as well as the time constant of the fast decay component changed consistently and reversibly with the membrane phospholipid PUFA composition. The current-voltage relationships, channel selectivity, rates of inactivation and recovery from inactivation did not change. Other parameters, such as time-to-peak and steady-state inactivation curves, have changed in EFA- and NFA-supplemented cultures and did not reverse completely. These findings demonstrate that the kinetics of INa can be modified by fatty acid supplementation. These effects can be correlated, in part, with alterations in plasma membrane phospholipid fatty acid composition.


Effects of HPA hormones on adapted lymphocyte responsiveness to repeated stress. Acute stress-induced immune alterations can result in adapted function with prolonged exposure to the same stressor. The present study was designed to evaluate the possible role of the hypothalamic-pituitary-adrenocortical (HPA) axis on the adaptation of spleen lymphocyte responsiveness to repeated stress. For this purpose, we selected a stressful protocol (aversive auditory stimulation) that induced an initial suppression (1 day), followed by a return to control values with repeated application (4 days), of mitogen-induced lymphocyte proliferation. Because rats exposed to 4 days of noise sessions show enhanced adrenocorticotropin (ACTH) and corticosterone levels, we tested the possibility that adaptation of lymphoproliferation by repeated stress was due to a desensitization of splenic lymphocytes to stress-released HPA hormones. The results showed that corticotropin-releasing factor (10(-9) M) and corticosterone (5 x 10(-8) and 10(-7) M), as well as dexamethasone (10(-8), 5 x 10(-8), and 10(-7) M), significantly suppressed lymphoproliferation from both control and stressed rats in a similar way. ACTH (10(-9) and 5 x 10(-9) M) did not significantly influence Concanavalin-A-stimulated spleen lymphocytes. These data indicate that adaptation of lymphocyte proliferation by repeated noise stress occurs without accompanying alterations in lymphocyte responsiveness to HPA hormones.


Effects of acute pentobarbital administration on GABAA receptor-regulated chloride uptake in rat brain synaptoneurosomes. Effects of acute pentobarbital administration on GABAA receptor-regulated muscimol-stimulated, pentobarbital-stimulated, or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied in the brains of Sprague-Dawley rats. Animals received sodium pentobarbital, 60 mg/kg IP, and cerebral cortical and cerebellar synaptoneurosomes were isolated at 10 min, 1 h, and when animals had awakened. The basal uptake of chloride was not changed in either cerebral cortex or cerebellum at different time periods after pentobarbital administration. Ten minutes after sodium pentobarbital administration, muscimol-stimulated chloride uptake was significantly reduced in cerebellum when the muscimol concentration was 2.5, 5, or 20 microM and in cerebral cortex when the concentration of muscimol was 5 or 10 microM (p less than 0.05, Duncan multiple-range test). One hour after pentobarbital administration or after animals had awakened, chloride uptake in brains from pentobarbital-treated animals was less at low concentration of muscimol (2.5 microM). No significant difference was found in either cerebral cortex or cerebellum in pentobarbital-(125-1,000 microM) stimulated or flunitrazepam-(2.5-20 microM) enhanced, muscimol-(3 microM) stimulated chloride uptake at different time periods after pentobarbital administration. Saline treatment had no effects on the basal or muscimol-stimulated chloride uptake in cerebellar synaptoneurosomes when compared with naive animals. The results demonstrate that GABAA receptor-regulated chloride uptake is decreased after acute pentobarbital administration, an effect that is reversible.


Paragigantocellularis stimulation induces beta-adrenergic hippocampal potentiation. The nucleus paragigantocellularis (PGi) is the major excitatory glutamatergic input to the locus coeruleus (LC). Glutamate activation of the LC has previously been shown to produce beta-adrenergic-dependent potentiation of the perforant path- (PP) evoked population spike in the dentate gyrus (DG). The present study asks if electrical stimulation of the PGi, by activating the LC endogenously, can produce a parallel beta-receptor-dependent potentiation of the PP-evoked population spike. An optimal interstimulus interval (ISI) was determined for PGi-PP stimulation in urethane-anesthetized rats and propranolol was used to assess the role of noradrenergic beta-receptors. PGi stimulation potentiated the PP-evoked population spike at an optimal ISI of 30 ms. The population synaptic response slope and spike latency were not affected. Propranolol blocked the PGi-induced potentiation, as would be expected for beta-receptor-dependent modulation. The parallels between PGi- and LC-induced effects on the PP-evoked population spike suggest PGi stimulation offers an alternate method of LC activation for studies of LC's role in behavior.


Regional and subcellular distribution of [125I]endothelin binding sites in rat brain. The binding of [125I]endothelin-1 (125I-ET-1) to membranes from whole rat brain, from individual brain regions, and derived from subcellular fractionation of whole rat brain was investigated. 125I-ET-1 binding to whole rat brain membranes was rapid, concentration-dependent, saturable, and characterized as irreversible because it was not displaced by unlabeled endothelin-1 (ET-1) and different concentrations of ligand produced, with time, a similar magnitude of binding. The maximum binding site capacity and second-order forward rate association constant of binding were 1,946 +/- 147 fm/mg protein and 5.53 +/- 1.72 x 10(6) M-1 s-1. Removal of either extramembranal calcium or membrane-bound calcium and calcium binding proteins did not affect the binding of 125I-ET-1 to whole rat brain membranes. The brain stem and cerebellum contained the highest levels of 125I-ET-1 binding sites, whereas the cerebral cortex, striatum, and hippocampus contained binding site levels three- to fourfold less. Subcellular fractionation of whole rat brain and subsequent analyses of the distribution of 125I-ET-1 binding demonstrated a twofold enrichment of binding sites in the synaptosomal fraction compared to the homogenate. The myelin fraction contained a similar density of binding sites compared to the homogenate, while the mitochondrial and microsomal fractions contained considerably less binding sites. The ribosomal fraction did not contain any 125I-ET-1 binding sites. The subcellular distribution of 125I-ET-1 binding sites did not correlate with the distribution of 5'-nucleotidase, cytochrome-C oxidase, phosphodiesterase, and alkaline phosphatase. Depletion of extracellular calcium increased 125I-ET-1 binding in the synaptosomal fraction but not in the myelin and mitochondrial fractions.(ABSTRACT TRUNCATED AT 250 WORDS)


NMDA and non-NMDA receptor antagonists inhibit photic induction of Fos protein in the hamster suprachiasmatic nucleus. We previously reported that systemic treatment with a noncompetitive antagonist affecting the NMDA subtype of excitatory amino acid (EAA) receptor, MK-801, inhibits photic induction of Fos-like immunoreactivity (Fos-lir) in the hamster suprachiasmatic nucleus (SCN). Because MK-801 blocks the Ca2+ channel associated with the NMDA receptor, it may also influence the activity of other transmitters acting through Ca2+ channels. To assess the specificity of these effects, we compared the effects on photic induction of Fos-lir of several treatments: central injection of a competitive NMDA antagonist, CPP; central injection of a non-NMDA antagonist, DNQX; and systemic injection of the non-competitive NMDA antagonist, ketamine. Fos-lir was induced in SCN cells of vehicle-injected hamsters exposed to a light pulse 4-5 h after dark onset. Pretreatment with CPP (greater than 2 nmoles) or ketamine (greater than 100 mg/kg) caused a dose-related inhibition of photic induction of Fos-lir in portions of the SCN. These treatments reduced Fos-lir mainly in the rostral SCN and ventrolateral, but not dorsolateral, portions of the caudal SCN. Pretreatment with DNQX (greater than 20 nmoles) also inhibited photic induction of Fos-lir in the same regions of the SCN. These results indicate that photic induction of Fos protein in a portion of the hamster SCN is regulated by both NMDA and non-NMDA types of EAA receptor.


[Exercise prescription for obese children]. Exercise prescription for obese children was formulated according to the characteristics of development of aerobic capacity of children of China and obese children as follows: intensity: 50% of VO2 duration: 1 hr/day, 12 weeks; frequency: 5 days per week; type of exercise: rhythmical and aerobic in nature such as running and various endurance game-type activity (football).


[Determination of renotropic activity in the sera of 35 uninephrectomy patients]. The renotropic growth factor (RGF) does increase in postuninephrectomy serum although the majority of studies were conducted in experiment. As regard to whether there is postnephrectomy RGF in human being, we have not hitherto reached a conclusion. By incorporating tritiated thymidine (3H-TdR) into primary human kidney cells in culture. We have determined renotropic activity in the sera of 35 uninephrectomy patients. The RGF was found increased in 68% and 76% patients at postnephrectomy days 2 and 5 respectively, showing an average increase of 14% and 33% as compared with that of preoperation level. i.e. The RGF does exist in the sera of uninephrectomy patients. Through multifactorial analysis, it revealed that renotropic activity was higher in male and uninephrectomy patient with better renal function. The same was also found in the patient who had not encountered side effect derived from blood transfusion and that the kidney lesion was benign in nature. Female or senile patient with deteriorated renal function or those with malignant lesion or transfusion side effect showed a lower renotropic activity. But no significant statistic difference was seen between the two categories.


[Determination of plasma level of platelet activating factor in cirrhotic patients and its relation to endotoxemia]. The plasma levels of platelet activating factor (PAF) were measured in 27 decompensated cirrhotic patients and 11 controls, using platelet aggregation bioassay. At the same time, the levels of endotoxin were also measured. The results showed that the levels of plasma PAF in decompensated cirrhotic patients were higher than those in the controls (5.34 +/- 1.94 ng/ml:1.18 +/- 0.24 ng/ml, P less than 0.01), and were higher in the cirrhotic patients with endotoxemia than in those without endotoxemia (6.19 +/- 2.21 ng/ml:3.89 +/- 0.92 ng/ml, P less than 0.01). The level of plasma PAF is related significantly to the level of endotoxin (r = 0.67, P less than 0.01)). We conclude that plasma PAF is increased in cirrhotic patients, especially in those with endotoxemia, and PAF could be involved in the pathophysiological changes in endotoxemia and other complications of cirrhosis.


[Radioimmunoassay and immunohistochemical study of myoglobin in neuromuscular diseases]. The localization of myoglobin (Mb) in skeletal muscle was studied by an immunoperoxidase technique and the serum Mb was measured by radioimmunoassay in 38 patients with various neuromuscular diseases. The positive rate of Mb immunoreactivity was significantly different between the muscular disorders and the motor neuron diseases. It was 44-77% in Duchenne dystrophy and polymyositis, and was 90-99% in motor neuron diseases. Serum myoglobin was markedly elevated in all the patients with muscular disorders, especially Duchenne dystrophy and polymyositis at the level of 329-330 ng/ml. It was found to increase slightly or normally in motor neuron diseases. Marked decrease or loss of Mb immunoreactivity was observed in muscle fibers with hyaline degeneration or floccular necrosis. It suggested that the elevation of serum Mb in patients with these diseases was ascribable to its leak through the injured plasma membrane of diseased muscle fibers or release from the necrotic muscle fibers.


[Relation between emphysema and diaphragmatic fatigue]. Emphysematous rats induced by elastase were divided into 5 groups: N (control), P1 (elastase for 3 mo.), P2 (elastase for 4.5 mo.; NS intraperitoneally for 1 mo. after 3.5 mo. elastase); A and C, aminophylline and ginseng preparation were respectively given in the same fashion as P2. The results were: 1. tension and endurance of diaphragm muscle decreased under low frequency stimulation in P2, while those no change in P1. DNA, RNA and ATPase increased, cross-sectional areas and glycogen decreased in P1, while ATPase and RNA reduced with increase of cross-sectional areas in P2; 2. in A, no change in tension, with enhanced endurance; 3. in C, tension increased with no change in endurance; 4. glycogen and cross-sectional areas increased in both A and C; 5. RNA and ATPase increased in C and no change in A.


[Role of Langerhans' cells against cervical human papilloma virus infection and development of cervical carcinoma]. Human papilloma virus (HPV) in the development of cervicitis and cervical carcinoma, and the role of Langerhans' cells (LC) against cervical HPV infection and development of cervical carcinoma were studied by immunohistochemistry and by DNA molecular hybridization techniques for the HPV type 6, 11, 16 and 18 DNA sequences. The total results were analysed and correlated. It was found that the positive rate of HPV type 6 and 11 in cervicitis was significantly higher than that in cervical carcinoma (P less than 0.01), and the positive rate of HPV type 16 and 18 was higher than that in cervicitis, but only the difference of HPV type 16 positive rate reaches significance level (P less than 0.01). The results also showed that the mean density of S100 + LC in cervical carcinoma specimens was significantly higher than that in cervicitis specimens (P less than 0.01). The results indicate that HPV type 6 and 11 are closely related with cervicitis, and HPV type 16 and 18 are closely related with the development of cervical carcinoma. LC plays an important role in attacking HPV infection of the cervix and the development of cervical carcinoma. The results also suggest that S100 + LC and OKT6 + LC are different subtypes of LC, and S100 + LC is an activated-subtype which plays a major role of immunosurveillance function.


[Magnesium sulfate in prevention of preterm labor]. To observe the efficacy of magnesium sulfate in the treatment of preterm labor, 65 uncomplicated cases of preterm labor between 28 and 36 weeks of gestation were studied prospectively during Sep. 1988-May 1991. They were divided into two groups randomly. 30 cases were treated with magnesium sulfate and 35 cases with barbiturates or bed rest. The prevention of delivery for at least 48 hours after the initiation of therapy was achieved in 23 of the 30 cases (76.67%) of the magnesium sulfate group and in 3 of the 35 cases (8.57%) in the control group. Delay of more than 7 days was achieved in 17 of the 30 cases (56.67%) and in 2 of the 35 cases (5.71%). The postponement of delivery between the two groups. There was highly significantly difference (P less than 0.01). There was a significant correlation of cervical dilation at the onset of treatment to success of controlling preterm labor. In the magnesium sulfate group, the mean magnesium level to achieve tocolysis was 2.8 +/- 0.35 mmol/L (mean +/- s). The side effects to the mothers, fetus, and the neonates were mild and not prominent.(ABSTRACT TRUNCATED AT 250 WORDS)


[Determination and clinical significance of lipoprotein(a) in cardio-cerebrovascular diseases]. We determined the level of lipoprotein (a) [Lp(a)] in 1,266 healthy persons and established the reference value of Lp(a) in various groups of age and sex. The analysis of lipids in myocardial infarction survivors and stroke survivors suggest that increased Lp(a) is an independent risk factor in cardio-cerebrovascular diseases. The characteristics of abnormal lipids in cardio-cerebrovascular diseases are discussed.


Physiology and pathophysiology of atrial natriuretic factor in lungs. OBJECTIVE AND DATA SOURCE: Experimental and clinical data reported in the international literature have been collected and critically reviewed to summarize knowledge of the role of atrial natriuretic factor (ANF) in lung physiology and pathophysiology. Lung contribution to circulating ANF concentration is modest, whereas its capability of degrading ANF is very high, the lung being one of the major sites of ANF catabolism. The impairment of ANF protease activity in lung tissue by hypoxia and pulmonary hypertension could be responsible for the increase in ANF plasma levels observed in several pulmonary pathological conditions. ANF-specific binding sites in lung are reportedly greater than in any other tissue. ANF induces a cGMP-mediated relaxation of central (rather than peripheral) bronchi. ANF bronchodilating effect has also been clinically demonstrated; eg, asthmatic patients show increased plasma ANF levels and exogenous ANF infusion provokes bronchial relaxation comparable with the salbutamol-induced effect. Moreover, ANF determines pulmonary artery vasodilation, thus contributing to improved pulmonary circulation. When pathophysiological levels are present in plasma, ANF influences pulmonary fluid regulation provoking protein mobilization from arteries to the alveolar space whereas ANF pharmacological concentrations re-equilibrate the transwall gradient. A remarkable enhancement of guanylate cyclase activity in lung tissue before hemodynamic modifications by both endogenous end exogenous ANF has been reported in pneumocytes of cardiomyopathic hamsters. On the other hand, ANF infusion provokes a reduction of pulmonary edema induced by pneumotoxic chemicals through a mechanism independent of the natriuretic/hypotensive action of the peptide and not mediated by cGMP. The modest amount of specific research on ANF effects on lung does not permit a final assessment of natriuretic peptides in pulmonary physiology and pathophysiology. In particular, further investigations are needed to determine the potential clinical relevance of ANF in asthma and pulmonary edema.


Transplant atherosclerosis: the clinical syndrome, pathogenesis and possible model of spontaneous atherosclerosis. To summarize the clinical syndrome of transplant atherosclerosis, including the clinical risk factors, pathological features and known pathophysiological mechanisms. The review also examines transplant atherosclerosis and naturally occurring atherosclerosis to delineate common pathophysiological mechanisms and to promote transplant atherosclerosis as a model for the more commonly occurring spontaneous process. Information gathered includes studies related to transplantation, endothelium, atherosclerosis, macrophages, lipoproteins and vascular smooth muscle. All studies cited were published prior to 1992. Transplant atherosclerosis appears to result from a 'response to injury' of the endothelium, similar to naturally occurring atherosclerosis. The injury is diffuse, chronic and related to immune and nonimmune factors. Cellular and antibody-mediated rejection, cytomegalovirus infection and cytokine-induced vessel wall cell proliferation provide potential targets for future pharmacological and biological therapy.


Pseudomyxoma peritonei of appendiceal origin. The Memorial Sloan-Kettering Cancer Center experience. Pseudomyxoma peritonei is a rare clinical entity in which the peritoneal surfaces and omentum are involved with diffuse gelatinous mucinous implants. It originates from ruptured mucinous tumors of the appendix or ovary. The authors examined the experience with 34 patients with pseudomyxoma peritonei seen at Memorial Sloan-Kettering Cancer Center from 1952-1989. Of these, 17 cases were identified to be of appendiceal origin. All patients underwent celiotomy and cytoreduction. The median survival time from diagnosis was 75 months. It was found that long-term survival can be achieved by operation alone. When conditions do recur, chemotherapy may be valuable. Pseudomyxoma peritonei of appendiceal origin is a rare low-grade malignancy. Initial treatment consists of cytoreduction in an attempt to render the patient locally disease-free. Long-term survival can be obtained by operation alone, even if gross disease is present at the end of the procedure. Systemic chemotherapy should be reserved for patients with proven recurrence.


A cholangiocellular carcinoma nude mouse strain showing histologic alteration from adenocarcinoma to squamous cell carcinoma. Adenosquamous carcinoma and squamous cell carcinoma (SCC) occur rarely in the liver compared with adenocarcinoma, and the histogenesis and biologic behaviors of these tumors remain unknown. The authors addressed these issues in the current article. A specimen aseptically obtained from the surgically resected cholangiocellular carcinoma (CCC) was cut into pieces and inoculated into the back of a nude mouse, bilaterally. The developed tumors were resected and serially transplanted into nude mice. The morphologic features and growth kinetics of the nude mouse tumors at different passages were compared. The authors established a new human CCC nude mouse strain, designated nuKMC-2, from a 64-year-old woman. The original tumor of the patient showed the features of moderately differentiated tubular adenocarcinoma with small sheet-like arrangement of polygonal cells. The initial tumor developed in a nude mouse showed morphologic features similar to the original tumor. With the serial transplantation to nude mice, the components of tubular adenocarcinoma diminished, and all of the nuKMC-2 was replaced by SCC. Doubling times of nuKMC-2 at the 5th and 11th passages were 9.9 and 7.4 days, respectively, which suggested that the tumor with squamous components were more aggressive biologically than the adenocarcinoma. The results suggested that adenosquamous carcinoma might be a transitional form from adenocarcinoma to SCC and that some of the primary hepatic SCC might originate from adenocarcinomas.


Sinonasal primitive neuroectodermal tumor arising in a long-term survivor of heritable unilateral retinoblastoma. Patients who survive retinoblastoma (RB) are at risk for having second nonocular tumors, usually osteosarcomas, which often are fatal. Such patients almost always have bilateral RB. This article reports a woman who, at the age of 1 year had been cured of a unilateral RB by radiation therapy and enucleation. Eighteen years later, she had a sinonasal small cell tumor that rapidly recurred and proved fatal 2 months after surgical debulking. The tumor was studied by immunohistochemistry and electron microscopic (EM) examination. It showed diffuse neuron-specific enolase staining, focal weak staining for chromogranin, synaptophysin, and Leu-7 monoclonal antibodies in paraffin-embedded, B5-fixed tissue (Great Lakes Diagnostics, Troy, MI). EM study showed an undifferentiated primitive neuroectodermal tumor with many polyribosomes, simple cell junctions, few microtubules, and rare dense core granules. The combined immunohistochemical, ultrastructural, and clinical features of the tumor were interpreted as a sinonasal primitive neuroectodermal tumor with early neuronal differentiation. The tumor was pathologically indistinguishable from poorly differentiated olfactory neuroblastoma (ONB) and Ewing sarcoma.


Ultrastructural and ultracytochemical differences between megakaryoblastic leukemia in children and adults. Analysis of 49 patients. Acute megakaryoblastic leukemia (AMKL) has two peaks in distribution of incidence (in adults and children 1 to 2 years of age) and is frequently seen in children with Down syndrome. The current study was undertaken to disclose whether there were any differences between these groups. Electron microscopic and ultrastructural cytochemical features of 49 children and adults with a AMKL or chronic myelogenous leukemia (CML) in megakaryoblastic crisis were compared. Blast cells from children with AMKL, including those with and without Down syndrome, had immature features lacking typical alpha granules and a demarcation membrane system (DMS). However, blast cells from patients with AMKL with Down syndrome had more theta, electron-lucent, and basophil-like granules, suggesting that the blast cells had more potential to differentiate into other cell lines than megakaryocytes. The AMKL blast cells of adult patients showed a higher percentage of platelet peroxidase (PPO) positivity than other subgroups, and they occasionally contained typical alpha granules and DMS. This indicated that the blast cells of adults with AMKL were more mature than those of children and CML in megakaryoblastic crisis. By electron microscopic analysis, leukemic megakaryoblasts differed between children with AMKL with and without Down syndrome, adults with AMKL, and patients with CML in megakaryoblastic crisis.


Regressing atypical histiocytosis, a regressing cutaneous phase of Ki-1-positive anaplastic large cell lymphoma. Immunocytochemical, nucleic acid, and cytogenetic studies of a new case in view of current opinion. Regressing atypical histiocytosis is a rare multifocal cutaneous tumor characterized by large, spontaneously regressing, ulcerating skin nodules. Although initially self-remitting, the condition may progress to systemic lymphoma. Using material from one patient, an attempt was made to clarify the nature of this condition with immunophenotyping, genotyping, and chromosome studies. Immunophenotyping studies indicated the condition was of T-cell lineage, although T-cell receptor gene studies showed polyclonal rearrangement. This case progressed to systemic lymphoma. The authors believe regressing atypical histiocytosis is a regressing phase of Ki-1-positive anaplastic large cell lymphoma of the skin.


Tendosynovial sarcoma. Clinicopathologic features, treatment, and prognosis. Clinicopathologic features, treatment, and results are reported for 95 tendosynovial sarcomas identified from a prospective sarcoma data base established at Memorial Sloan-Kettering Cancer Center (MSKCC) in 1982. Eighty-five patients had definitive treatment of the primary tumor at MSKCC: Epithelial sarcomas were excluded. The local recurrence rate was 18%. Survival rates were not influenced by either the method of local treatment or the use of chemotherapy. The overall actuarial survival rate was 59% at 5 years (median, 92 months). For 25 patients who had resection of pulmonary metastases, the 5-year actuarial survival rate was 43% (median, 22 months) from the time of first thoracotomy. Sex, age, tumor site, and histologic subtype (monophasic versus biphasic) were not significant predictors of survival rates. Regional lymph node metastasis occurred in 3%. The incidence of distant metastasis was 49%, with an associated 5-year survival rate of 31% (median, 33 months). Tumor size was a highly significant prognostic feature, with 5-year survival rates of 86% for patients with tumors smaller than 5 cm and 22% for those with tumors larger than 10 cm (P less than 0.00001). Improvement of treatment results for tendosynovial sarcoma will require effective new systemic adjuvant therapeutic strategies.


Epstein-Barr virus-associated primary B-cell lymphoproliferative disorder of the cerebellum in an immune competent man. The role of the Epstein-Barr virus (EBV) in lymphoproliferative lesions has been widely accepted. Most of these lesions occur in patients who have deficiencies in their immune status. Lymphomatoid granulomatosis (LG) is a lymphoproliferative disorder originally characterized as an angiocentric, necrotizing, pleomorphic infiltrate of mononuclear cells. The etiology of LG is unknown. It was originally hypothesized that LG may represent an unusual lymphoid response to an infective organism, possibly EBV. Tissues from a previously healthy 60-year-old, healthy white man with primary cerebellar lymphomatoid granulomatosis were examined for the presence of EBV by nucleic acid hybridization. The original LG lesion was a polyclonal B-cell proliferation that contained detectable amounts of EBV. Peripheral blood leukocytes were negative for EBV by the same assay. After an 18-month remission, a tumor reappeared near the site of the primary lesion, which had the histologic appearance of a lymphoma. The cells showed restricted clonality and contained a similar amount of EBV-related DNA as the original lesion. Peripheral blood leukocytes at the time of recurrence were negative for EBV. The patient died approximately 2 months after the recurrent tumor was detected. This case demonstrated the development of a primary cerebellar B-cell lymphoproliferative disorder, histologically identical to lymphomatoid granulomatosis, that transformed into a lymphoma. The original tumor and the subsequent lymphoma contained, on average, several copies of EBV-related DNA per cell. Despite an extensive survey of the patient, no immune deficit was detected. Interpretation of the literature with the results of this case suggest that this instance of primary cerebellar LG arose as a consequence of an unusual EBV-associated B-cell lymphoproliferation. It is suggested that EBV may be a significant factor in the initiation of the abnormal proliferations of T-cells or B-cells reported in this disorder.


Histopathologic grading and DNA ploidy in relation to survival among 206 adult astrocytic tumor patients. The authors studied the benefit of performing histopathologic grading and DNA ploidy characterization with respect to patient survival in a series of 206 astrocytomas (AST) for which they obtained 134 complete clinical follow-ups. The material analyzed came from archival material, i.e., formalin-fixed paraffin-embedded tissues. DNA ploidy was assessed by means of a cell image processor computing the integrated optical density (IOD) on Feulgen-stained nuclei. Results showed that histopathologic diagnosis in three grades, i.e., AST, anaplastic astrocytoma (ANA), and glioblastoma multiforme (GBM), had a significant prognostic value. Patients with AST showed a mean survival time (between histopathologic diagnosis and death) of more than 36 +/- 6 months (AST versus ANA or GBM) (P less than 0.001). Patients with ANA and GBM showed a mean survival time of 15 +/- 2 and 10 +/- 1 months, respectively, (ANA versus GBM) (P less than 0.05). Patient age strongly correlated with survival. Patients younger than 40 years of age had a mean survival time of 20 +/- 4 months. Patients between 41 and 60 years of age had a mean survival time of 12 +/- 2 months, and patients older than 60 years of age had a mean survival time of 11 +/- 1 months. Considering DNA ploidy characterization, the authors noticed that aneuploid ANA (DNA index [DI] more than 1.30) were associated with a significantly higher mean patient survival time compared with that associated with euploid ANA. In contrast, the authors did not find this in either of the groups with AST and GBM. Recognizing six DNA histogram types (diploid, triploid, tetraploid, hyperdiploid, hypertriploid, and polymorphic), the authors observed that hypertriploid tumors were associated with greater patient survival compared with what happened in the cases of the five other DNA histogram types. This was true with respect to the three AST histopathologic types. Thus, DNA ploidy determination seemed helpful in characterizing aggressiveness in adult AST.


Endocrine secretory granules and crystals in the syncytiotrophoblast. Large membrane-bound crystalline inclusions were seen on electron microscopic examination of the syncytiotrophoblast from the placenta of a HIV infected patient. The syncytium displayed abundant secretory granules (180-240 nm in diameter), with electron-dense centers (i.e., bull's eye granules or targetoid granules) which occasionally showed partial crystallization. Some crystals exhibited a periodicity of 9-10 nm. Electron-dense material was also seen in distended cisternae of the rough endoplasmic reticulum (RER). These granules correspond presumably to human placental lactogen (HPL) secretion, as seen on light microscopic immunoperoxidase stains. The morphological evidence is consistent with the idea that the crystals represent crystalline accumulation of secretory product (HPL), thus forming an analogy with crystal formation in other types of secretory cells. Ribosome-lamella complexes were seen in the many pericytes present in the capillaries of chorionic villi.


Modulation of the cell cycle-dependent cytotoxicity of adriamycin and 4-hydroperoxycyclophosphamide by novobiocin, an inhibitor of mammalian topoisomerase II. Centrifugal elutriation was used to obtain synchronized cell populations in various cell cycle phases without prior growth-perturbing manipulation. Treatment of these subpopulations with novobiocin (NOVO), a putative inhibitor of the mammalian topoisomerase II enzyme, revealed a unique cell cycle phase-dependent cytotoxicity for this agent. At a concentration of 0.3 mM, NOVO was cytotoxic only to a specific cell subpopulation in the G1-S phase boundary. Cells in other cell cycle phases were completely unaffected. Additionally, S and G2M phase cells progressed through the cell cycle relatively unaffected by NOVO but were blocked at the G1-S boundary. NOVO treatment protected tumor cells from Adriamycin (ADR)-induced lethality but sensitized them to the toxic action of 4-hydroperoxycyclophosphamide, and alkylating agent. These opposing effects of NOVO were demonstrated in all of the four tumor cell lines investigated: A431 and HEp3 (derived from human squamous cell carcinomas); MLS, a human ovarian cancer cell line; and a Chinese hamster ovary cell line. The degree of protection against ADR was the greatest for S-phase cells, intermediate for cells in early G1 and M phases, and the least for late G1 cells. This cell cycle-dependent protection by NOVO, which is identical to the cell cycle-dependent cytotoxicity of ADR, was consistent with the idea that NOVO interfered directly with the cell-killing mechanism of ADR. In contrast, even though the cytotoxic activity of 4-hydroperoxycyclophosphamide exhibited significant cell cycle dependency, NOVO enhanced 4-hydroperoxycyclophosphamide lethality equally for all cell cycle phases.


Antitumor activity of magainin analogues against human lung cancer cell lines. Magainin 1 and magainin 2, originally isolated from African clawed frog Xenopus laevis skin, inhibit the growth of bacteria and fungi. Synthetic magainin A (MAG A) and magainin G (MAG G) are more potent against bacteria and protozoa. In order to determine the antitumor activity of these analogues, we have tested these two analogues against six small cell lung cancer (SCLC) cell lines NCI-H82, NCI-H526, NCI-H678, NCI-H735, NCI-H841, and NCI-H889, which were known to differ by more than 10-fold in their sensitivity to different chemotherapeutic agents, and four normal human fibroblast cell lines. Semiautomated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays of the six SCLC cell lines revealed average concentrations producing 50% inhibition (IC50) values of 2.6 microM (range, 0.49-9.30 microM) for cisplatin, 2.5 microM (range, 0.39-6.00 microM) for etoposide, and 138.8 nM (range, 55.0-450.0 nM) for doxorubicin. The average IC50 of MAG A was 8.64 microM (range, 6.23-11.7 microM) and that of MAG G was 8.82 microM (range, 4.44-12.5 microM) against the SCLC cell lines. Despite a 10-fold difference in sensitivity to standard chemotherapeutic agents, the IC50 of MAG A and MAG G differs by less than 3-fold. The average IC50 against four normal human fibroblast cell lines was 21.1 microM (range, 12.7-25.6 microM) for MAG A and 29.2 microM (range, 21.3-34.8 microM) for MAG G. Combined exposure to the IC50 concentration of MAG A or MAG G plus IC50 of etoposide or cisplatin decreased the percentage of surviving SCLC cells to 29.0% (range, 26.1-31.7%). MAG A or MAG G had an additive effect when used with standard chemotherapeutic agents. These data suggest that MAG A and MAG G have in vitro antitumor activity against SCLC cell lines.


Changes in rectal epithelial cell proliferation and intestinal bile acids after subtotal colectomy in familial adenomatous polyposis. Subtotal colectomy and ileorectal anastomosis in familial adenomatous polyposis patients can induce temporary regression of adenomas in the rectum. The mechanism for this phenomenon is unclear. We evaluated the effect of colectomy on rectal mucosal proliferation, in relation to changes in bile acid metabolism. Four familial adenomatous polyposis patients were studied before and 3-6 months after surgery, and eight others 7-22 years postoperatively. Within 6 months after surgery, the size of the proliferative zone of the colonic crypts was found to be reduced (P less than 0.05). The proliferative activity of total colonic crypts was not affected within this period. More than 7 years postoperatively, increased cell proliferation of total crypts (P less than 0.02), as well as mid (P less than 0.05) and basal (P less than 0.05) crypt compartments, were observed compared to shortly after colectomy. In duodenal bile, deoxycholic acid was absent shortly after operation, whereas several years after operation only a small fraction (2%) was present. Fecal secondary bile acid excretion diminished after colectomy and did not change several years postoperatively. In postoperative stools only, small proportions of ursocholic and ursodeoxycholic acids (about 5% each) were consistently found. As subtotal colectomy causes a temporary decrease in the length of the proliferative zone of rectal crypts toward a normal pattern, this may explain regression of rectal polyps. This temporary effect may be mediated, at least in part, by decreased amounts of cytotoxic secondary bile acids in the rectal lumen.


Multiple biological markers in germ cell tumor patients treated with platinum-based chemotherapy. Blood samples from 36 germ cell tumor patients receiving chemotherapy with either cisplatin or carboplatin in combination with other drugs [etoposide or vinblastine, cyclophosphamide, dactinomycin, and bleomycin (VAB-6)] were analyzed for the presence of 7 different biological markers. The biomarkers included platinum-protein adducts, platinum-DNA adducts, sister chromatid exchange (SCE), micronuclei (MN), and somatic gene mutation at the hypoxanthine phosphoribosyl transferase (HPRT) locus and the glycophorin A (GPA) loci (NO and NN). Patients were asked to donate 9 serial blood samples: a pretreatment sample followed by another drawn 12-24 h after each of four cycles of treatment and a final sample provided 3-6 months after the last cycle. Most individuals gave 7-8 samples; 7 individuals donated all 9. Because of limited amounts of cells in some cases, it was not possible to carry out all 7 assays on every sample. Pt-protein adducts, Pt-DNA adducts, and SCE showed a direct and consistent effect of treatment and were very highly correlated. A significant correlation was also seen between both Pt-protein and Pt-DNA adducts and HPRT mutation. All of the posttreatment samples were significantly elevated compared to the baseline sample. These markers also remained elevated 3-6 months after the end of treatment. By contrast, MN, HPRT mutation, and GPA mutation (both NO and NN variants) showed varying patterns of dose response, probably reflective of the differing biology of these markers scored in lymphocytes (MN and HPRT) and erythrocytes (GPA). MN were significantly elevated in the posttreatment samples drawn at cycles 2 and 3. Although induction of HPRT mutation was only of marginal significance, results here are for the mutant frequency determination assay only. In progress is the potentially more informative analysis of the type of mutations by Southern blot and the sequencing of mutations to look for characteristic mutational spectra. The GPA assay showed a significant increase over baseline in samples drawn after cycles 3 and 4 (NO variants) and after cycles 2, 3, and 4 (NN variants). The level of GPA mutation (both NO and NN variants) was clearly elevated even 3-6 months after the last cycle of chemotherapy. Correlations were seen between HPRT and MN as well as between GPA NO and GPA NN variants. Analysis of biomarkers by treatment group does not reveal a consistent pattern or trend across all cycles.(ABSTRACT TRUNCATED AT 400 WORDS)


Inhibition of hepatocyte DNA synthesis by transforming growth factor beta 1 and cyclic AMP: effect immediately before the G1/S border. Previous studies have shown that both transforming growth factor beta (TGF-beta) and cyclic AMP (cAMP) inhibit hepatocyte DNA synthesis. While cAMP (in addition to being stimulatory in G0/early G1) exerts its inhibition on hepatocytes late in G1, the point where TGF-beta inhibits has not been precisely defined. We have now examined further the inhibitory effects of cAMP and TGF-beta 1 on DNA synthesis in primary rat hepatocyte cultures and, in particular, tried to determine where in the prereplicative period the cells are sensitive to these agents. Although a transient exposure to TGF-beta 1 (but not glucagon) during the first hours of the cell culturing led to inhibition of DNA synthesis, the cells were more sensitive at a point late in G1, where they also were inhibited by cAMP. Thus, exposure to TGF-beta 1, glucagon, or the cAMP analogue 8-chlorophenylthio-cAMP at a time when there was a continuous recruitment of cells to S phase strongly decreased the rate of S-phase entry. For both TGF-beta 1 and cAMP the inhibition was established within 1-2 h, the lag time being indistinguishable for the two agents. No evidence was found for a synergism between TGF-beta 1 and cAMP. Treatment with TGF-beta 1 did not detectably alter basal or glucagon-stimulated cAMP concentrations. The results suggest that in hepatocytes there is a process immediately before the G1/S border which is sensitive to both TGF-beta 1 and cAMP and which appears to represent a major point of inhibition.


Mutation pattern of the p53 gene as a diagnostic marker for multiple hepatocellular carcinoma. Hepatocellular carcinoma, sometimes shows multiple tumor nodules, therefore poses a problem of differential diagnosis between cancers of multifocal and those of metastatic origin. Conventionally, pathological criteria have been used for this purpose, but these are largely subjective. In order to facilitate more objective differential diagnosis of multiple hepatocellular carcinoma, we used the pattern of mutation of the p53 gene as a marker for each tumor nodule. We studied 58 nodules from 26 cases of multiple hepatocellular carcinoma using polymerase chain reaction-single strand conformation polymorphism analysis, a simple method for detecting mutations. p53 gene mutations were detected in 65% (17 of 26) of cases. The internodule mutation patterns were heterogeneous in 11 cases and homogeneous in 6, enabling a multifocal origin to be diagnosed in the former and a metastatic origin in the latter at the genetic level. Moreover, the origin of recurrent tumors was determined from the mutation pattern. It is concluded that analysis of p53 mutations seems to be useful for differentiating the origin of multiple cancers, since the information it yields is essentially objective.


Characterization of the PML-RAR alpha chimeric product of the acute promyelocytic leukemia-specific t(15;17) translocation. The acute promyelocytic leukemia 15;17 chromosomal translocation fuses the PML gene to the RAR alpha locus. The resulting chimeric gene encodes for a putative PML-RAR alpha fusion protein. PML is a putative transcriptional factor and RAR alpha is one of the nuclear retinoic acid receptors through which retinoic acid regulates gene expression. In this study, we investigated the retinoid binding and biochemical properties of the PML-RAR alpha protein by size exclusion high-performance liquid chromatography and immunoblot analysis and compared them with those of normal RAR alpha. The introduction of the expression vector PSG5/PML-RAR alpha into COS-1 cells led to high levels of expression of the PML-RAR alpha fusion protein. This protein was primarily localized in the nucleus and bound retinoids with the same affinity and specificity as the wild type RAR alpha receptor. The PML-RAR alpha fusion protein, but not the RAR alpha, was found in high molecular weight complexes with either itself or other nuclear factors. In the acute promyelocytic leukemia-derived cell line NB4, which contains the t(15;17) chromosomal marker, the PML-RAR alpha product was also found as a high molecular complex. The interaction of the PML-RAR alpha with itself or with other nuclear proteins may be important in understanding the role of the PML-RAR alpha fusion protein in promyelocytic leukemogenesis.


Gastrin-releasing peptide: in vivo and in vitro growth effects on an acinar pancreatic carcinoma. The mammalian gastrin-releasing-peptide (GRP) and its structural amphibian analogue, bombesin, are known to be trophic factors for the normal exocrine pancreas. This work investigates the possible role of GRP in the growth of an acinar pancreatic cancer transplanted to the rat and in primary tumor cell cultures. Moreover, this adenocarcinoma was tested for its content of specific bombesin/GRP receptors by using autoradiographic technics and in vitro binding assays with tumor cells. In Lewis rats bearing the pancreatic carcinoma transplanted s.c. in the scapular region, chronic administration of GRP at the dose 30 micrograms/kg/day for 15 successive days significantly increased the tumor volume, the final tumor weight, and amylase, protein, RNA and DNA contents. Autoradiographic studies showed that tumor tissue was GRP receptor positive with a high density. The biochemical characterization indicated that receptor positive tumor tissue had saturable and high affinity receptors with pharmacological specificity for GRP and its bioactive analogues. In primary tumor cell cultures, GRP increased the incorporation of [3H] thymidine in DNA in a dose- and time-dependent manner. There was a good correlation between the ability of GRP and its COOH terminal analogues to elicit DNA synthesis and their affinity for 125I-GRP binding sites. These results from in vivo and in vitro experiments demonstrated that GRP induces growth of pancreatic carcinoma by acting directly on specific membrane receptors present on the tumor cells.


SAS amplification in soft tissue sarcomas. Gene amplification is an important mechanism of increased gene expression in a number of human solid tumors. We have recently identified and cloned sequences from a novel DNA amplification unit in malignant fibrous histiocytoma. The amplified sequences are derived from chromosome 12q13-14 and encode a gene designated SAS (sarcoma amplified sequence). In the present study, a series of soft tissue sarcomas was studied to characterize further the phenomenon of SAS amplification. Seven of 22 (32%) malignant fibrous histiocytomas and three liposarcomas contained SAS amplification. Strikingly, all of the tumors with SAS amplification occurred in central sites (i.e., in the abdominal or inguinal regions) rather than in the extremities (i.e., in the arms of legs). These observations demonstrate that SAS amplification occurs with a significant frequency in mesenchymal tumors and is particularly associated with abdominal disease.


Oncogene complementation in fetal brain transplants. Using a neural transplantation model and retrovirus-mediated gene transfer, we have introduced the oncogenes v-Ha-ras and v-myc into the developing rat brain. Upon insertion of a construct encoding v-Ha-ras and the Escherichia coli beta-galactosidase marker gene, the retroviral vector was found to be expressed in neurons, astrocytes, and endothelial cells of the graft. After latency periods of several months, fascicular neoplasms with expression of S-100 protein were observed in 50% of the transplants. The foreign genes were shown to be highly expressed in the tumors and in intact donor cells, by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry, indicating that an activated Ha-ras oncogene has the potential to initiate neoplastic transformation of glial cells. Introduction of the v-myc oncogene into 15 grafts resulted in only a single primitive neuroectodermal tumor. However, simultaneous expression of the v-Ha-ras and v-myc genes yielded highly malignant, polyclonal neoplasms in all recipient animals, as early as 13 days after transplantation, from which cell lines could be easily derived. In addition, neoplastic transformation was also observed in vitro following introduction of ras and myc into embryonic forebrain cultures and into newborn cerebellar cultures. These data indicate a powerful complementary transforming effect of ras and myc on neural progenitors in vivo and in vitro. Coexpression of ras and myc may, therefore, provide a highly efficient tool for transforming neural precursor cells in distinct segments of the central nervous system at different stages of development.


UDP-N-acetylhexosamine modulation by glucosamine and uridine in NCI N-417 variant small cell lung cancer cells: 31P nuclear magnetic resonance results. Small cell lung cancer (SCLC) occurs as two neuroendocrine subtypes, SCLC-C (classic) and SCLC-V (variant). One reported difference is elevated levels of diphosphodiesters (DPDE) in the more differentiated SCLC-C subtype. DPDE have been identified as primarily UDP-N-acetylhexosamines (UDP-NAH) in a variety of tumors, and changes in DPDE levels have been observed during experiments designed to induce cell differentiation. UDP-NAH synthesis is controlled by negative feedback regulation of glutamine:fructose-6-P amidotransferase (EC 2.6.1.16), which can be circumvented by glucosamine. Using 31P nuclear magnetic resonance analysis of extracts and perfused cells, we have identified UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine as the primary metabolites in the DPDE spectral region of SCLC-V N-417 cells. Glucosamine addition causes a rapid increase in UDP-NAH levels. At glucosamine: glucose ratios of 1:1 and 10:1 formation of the UDP-NAH intermediates N-acetylglucosamine 6-phosphate and UDP-N-acetylglucosamine 1-phosphate is also observed, indicating UTP limitation. Subsequent uridine addition results in depletion of the intermediates and increased UDP-NAH formation. Moreover, N-417 cells retain the capacity to rapidly convert uridine to UTP despite low ATP and phosphocreatine levels. This expansion of the uridine pool may represent an additional metabolic reserve not yet addressed in the design of therapy options.


Loss of expression of the DCC gene during progression of colorectal carcinomas in familial adenomatous polyposis and non-familial adenomatous polyposis patients. We have previously observed that the frequency of loss of heterozygosity (LOH) on chromosome 18q was low in adenomas and intramucosal carcinomas, whereas invasive carcinomas exhibited a high frequency in familial adenomatous polyposis patients (M. Miyaki et al., Cancer Res., 50: 7166-7173, 1990). In the present study, LOH at the DCC locus on chromosome 18q and the expression of DCC gene into mRNA were analyzed in colorectal tumors with distinct histopathological types. The carcinomas that showed 18q LOH also lost the DCC locus. The expression of DCC gene into mRNA was examined at the level of 233-base pair fragments of nucleotide 986-1218 in DCC complementary DNA. In a moderate-to-severe adenoma, 5 carcinoma-in-adenomas, and 4 intramucosal carcinomas, the level of expression was as high as in normal colorectal mucosa, whereas it was greatly reduced or not detectable in most (13 of 16) invasive carcinomas. Among these invasive carcinomas, 7 of 11 showed 18q LOH, but 4 showed no LOH. These results suggest that the DCC gene is included in the allelic deletion on chromosome 18q, and that the progression of colorectal carcinoma from early stage to advanced stage accompanies the inactivation of the DCC gene through LOH and other mechanisms.


Retinoid antagonism of estrogen-responsive transforming growth factor alpha and pS2 gene expression in breast carcinoma cells. Exposure of MCF-7 breast carcinoma cells to estradiol results in an increase in transforming growth factor alpha (TGF-alpha) synthesis and secretion. Since TGF-alpha is a potent inducer of proliferation in MCF-7 cells, the increase in TGF-alpha production by estradiol is thought to play an important role in the estrogen stimulation of growth of these cells. Retinoic acid inhibits the proliferation of MCF-7 cells and antagonizes the estrogen stimulation of growth. Addition of retinoic acid resulted in a greater than 70% inhibition of estradiol-induced TGF-alpha synthesis and secretion in MCF-7 cells. The increase in TGF-alpha mRNA expression by estradiol was also inhibited by exposure of the cells to retinoic acid. Pretreatment of the cells with retinoic acid for 24 or 72 h caused more than 50 and 90% inhibition, respectively, of the estradiol-enhanced expression of TGF-alpha mRNA. Expression of pS2 mRNA in MCF-7 cells was stimulated approximately 8-fold by estradiol. Retinoic acid treatment suppressed by greater than 80% both the basal and estradiol-induced pS2 mRNA expression. Retinoic acid modulation of the estrogen receptor gene mRNA was not responsible for the retinoic acid inhibition of the stimulation of pS2 and TGF-alpha gene expression by estradiol, since estrogen receptor gene expression was increased rather than decreased in the presence of retinoic acid. The nuclear retinoic acid receptors alpha and gamma mRNA were expressed in MCF-7 cells and its retinoic acid-resistant derivative RROI. Addition of estradiol to MCF-7 cells resulted in a decreased expression of retinoic acid receptor gamma mRNA; this reduction is prevented by the presence of retinoic acid. These results indicate that retinoic acid can inhibit estradiol-induced TGF-alpha and pS2 mRNA expression in MCF-7 cells. The suppression of TGF-alpha expression may represent one possible mechanism by which retinoic acid antagonizes the stimulation of MCF-7 proliferation by estradiol.


Genetic changes of both p53 alleles associated with the conversion from colorectal adenoma to early carcinoma in familial adenomatous polyposis and non-familial adenomatous polyposis patients. Mutation and loss of heterozygosity (LOH) in the p53 gene were analyzed in 274 colorectal tumors of 4 histopathological grades. Among 160 tumors from 40 familial adenomatous polyposis patients, none of 58 adenomas with moderate dysplasia had p53 mutations, whereas 8% (3 of 37) of severe adenomas, 15% (6 of 40) of intramucosal carcinomas, and 40% (10 of 25) of invasive carcinomas had p53 mutations. Only 3% (1 of 33) of severe adenomas showed both mutation and LOH, while 25% (6 of 24) of intramucosal carcinomas and 40% (10 of 25) of invasive carcinomas had both mutation and LOH. All intramucosal and invasive carcinomas that had mutations lost the other allele of the p53 gene. In 114 tumors from 86 non-familial adenomatous polyposis patients, similar results were obtained; no adenoma showed both mutation and LOH, but both alterations occurred in intramucosal and invasive carcinoma. As regards specificity in 56 mutations detected in the present study, the frequently affected codons were codons 175, 238, 245, 248, 273, and 282, 4 of these amino acids being arginine, and 72% (39 of 54) of all mutations were GC to AT transition. Although expression into p53 polyadenylated RNA was high in every invasive carcinoma irrespective of the presence of mutation or LOH, there was a correlation between mutation and protein level; immunostaining of p53 protein was negative in almost all adenomas, but it was positive in 86% of invasive carcinomas exhibiting p53 mutation. These data suggest that genetic changes on both alleles of the p53 gene through mutation and LOH, which result in abnormal protein accumulation, are involved in the conversion of adenoma to early carcinoma. Also, carcinoma cells with p53 mutations existing within adenoma tissues are detectable by immunostaining, even in formalin-fixed, paraffin-embedded specimens.


Transforming growth factor beta 1 induces apoptotic cell death in cultured human gastric carcinoma cells. Transforming growth factor beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types, including tumor cells. We recently have reported the establishment and characterization of two human gastric scirrhous carcinoma cell lines, HSC-39 and HSC-43. Here we examined the effect of TGF-beta 1 on the growth of these lines as compared to five other human gastric adenocarcinoma cell lines. Proliferation of HSC-39 and HSC-43 cells was strongly inhibited by TGF-beta 1, whereas the other gastric adenocarcinoma cell lines were unresponsive to TGF-beta 1. Both HSC-39 and HSC-43 cells gradually lost viability following exposure to TGF-beta 1. This response was dose dependent up to 4 ng/ml. When TGF-beta 1 was removed, the cells failed to exhibit regrowth, indicating an irreversible growth-inhibitory effect of this agent, leading to cell death. DNA fragments were observed consisting of multimers of approximately 180 base pairs 24 h after TGF-beta 1 treatment. The chromatin condensation of each cell line was confirmed by Hoechst 33258 fluorochrome staining. Ultrastructurally, condensed and fragmented nuclei were observed in TGF-beta 1-treated cells. These features are generally associated with apoptotic processes. Both cell death and DNA fragmentation were partially inhibited by cycloheximide, suggesting the requirement for new protein synthesis. Our results suggest that TGF-beta 1 induces cell death in human gastric scirrhous carcinoma cells in vitro which is mediated by activation of a signal transduction pathway for apoptosis.


Abundant expression of immunoreactive endothelin 1 in mammary phyllodes tumor: possible paracrine role of endothelin 1 in the growth of stromal cells in phyllodes tumor. Immunoreactive endothelin 1 (irET-1) concentrations were measured in extracts prepared from 4 phyllodes tumors and 14 fibroadenomas. irET-1 was detectable in all tissue extracts by specific radioimmunoassay, and the mean concentration of irET-1 was 18-fold and 27-fold higher in tissue extracts from phyllodes tumors than in those from intracanalicular fibroadenomas and pericanalicular fibroadenomas, respectively. Reverse-phase high-performance liquid chromatography coupled with radioimmunoassay in the extracts from phyllodes tumors revealed one major irET-1 component corresponding to human standard ET-1. Furthermore, immunocytochemical staining for ET-1 revealed that numerous ET-1-immunoreactive cells were seen in the epithelial cells but not in the stromal cells, suggesting that ET-1 is synthesized by the epithelial component of phyllodes tumors. A possible paracrine role of ET-1 in the growth of this rare tumor which is characterized by its prominent stromal cellularity is discussed.


Correlation between the location of germ-line mutations in the APC gene and the number of colorectal polyps in familial adenomatous polyposis patients. Recently we have isolated the adenomatous polyposis coli (APC) gene which causes familial adenomatous polyposis (FAP), and its germ-line mutations in a substantial number of FAP patients have been identified. On the basis of this information, we compared the location of germ-line mutations in the APC gene in 22 unrelated patients (12 of whom have been reported previously) with the number of colorectal polyps developed in FAP patients; 17 were sparse types and five were profuse types. All but one of the mutations were considered to cause truncation of the gene product by frame-shift due to deletion (14 cases) or nonsense mutation (seven cases). The location of the germ-line mutations seems to correlate with the two clinical types; germ-line mutations in five FAP patients with profuse polyps were observed between codon 1250 and codon 1464, whereas mutations in 17 FAP patients with fewer polyps were observed in the other regions of the APC gene. The result suggests that the number of colorectal polyps in FAP patients may be associated with a difference in the stability or biological function of the truncated APC protein.


The periodate oxidation of sucrose in aqueous N,N-dimethylformamide. Sucrose has been oxidized with sodium periodate in 0-50% aqueous N,N-dimethylformamide (DMF). In 50% aqueous DMF the reaction is selective for the glucose ring, yielding a dialdehyde. The increased selectivity is not due to conformational factors but is ascribed to the dissociation of water from cyclic periodate ester species which makes the reaction via the acyclic ester on fructose unfavourable.


Modulation of connexon densities in gap junctions of horizontal cell perikarya and axon terminals in fish retina: effects of light/dark cycles, interruption of the optic nerve and application of dopamine. In the fish retina, connexon densities of gap junctions in the outer horizontal cells are modulated in response to different light or dark adaptation times and wavelengths. We have examined whether the connexon density is a suitable parameter of gap junction coupling under in situ conditions. Short-term light adaptation evoked low connexon densities, regardless of whether white or red light was used. Short-term dark adaptation evoked high connexon densities; this was more pronounced in the axon terminal than in perikaryal gap junctions. Under a 12 h red light/12 h dark cycle, a significant difference in connexon densities between the light and the dark period could be established in the gap junctions of the perikarya and axon terminals. Under a white light/dark cycle, only the gap junctions of axon terminals showed a significant difference. Crushing of the optic nerve resulted in an increase in connexon densities; this was more pronounced in axon terminals than in perikarya. Dopamine injected into the right eye of white-light-adapted animals had no effect. However, dopamine prevented the effect of optic-nerve crushing on connexon density. The reaction of axon-terminal gap junctions to different conditions thus resembles that of perikaryal gap junctions, but is more intense. Axon terminals are therefore thought to play an important role in the adaptation process.


Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs. 67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and -4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.


Interaction of the yeast pleiotropic drug resistance genes PDR1 and PDR5. The network of genes which mediates multiple drug resistance in yeast includes, among others, the PDR1 gene, which encodes a putative regulator of gene expression, and PDR5, a locus whose amplification leads to resistance. We demonstrate that disruption of PDR5 causes marked hypersensitivity not only to cycloheximide but also to sulphometuron methyl and the mitochondrial inhibitors chloramphenicol, lincomycin, erythromycin and antimycin. Genetic analysis of double mutants containing an insertion in PDR5 (pdr5:Tn5), which renders cells hypersensitive to cycloheximide, and a pdr1 mutation, which confers resistance to this inhibitor, indicates that the expression of resistance requires a functional PDR5 gene. The same interdependency is observed for chloramphenicol, but not for oligomycin, lincomycin, erythromycin or sulphometuron methyl. Northern analysis of PDR1 and PDR5 transcripts reveals that the 5.2 kbp PDR5 transcript is overexpressed in pdr1 (resistant) mutants, but underexpressed in a disruption of PDR1. These observations provide strong experimental support for our former proposal that the PDR5 gene is a target for regulation by the PDR1 gene product.


Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae. Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glycerol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.


The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning, physical mapping and gene location. The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80,500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.


[A study on the morphogenesis, tumor marker and DNA content in early gastric signet ring cell carcinoma]. From the study of 20 specimens of micro-signet ring cell carcinoma of stomach, it was noticed that fusiform epithelioid or round monocytoid carcinoma cells were usually found prior to the appearance of signet ring cells. Therefore, from the point of view in morphogenesis, cells of this tumor might be considered better to be differentiated into two stages--early and late stage, which not only represented a morphological evolution of these carcinoma cells, but also expressed their biological characteristics, as it was shown that the expressions of associated antigen to MCab MG-7 and receptors of PNA, PHA reached the highest degree in the monocytoid carcinoma cells, but was markedly attenuated in the signet ring cells and the difference present was 20 times quantitatively additionally, the nuclear DNA content was increased significantly in cancer cells of the early stage, especially the monocytoid cells, with a pattern of aneuploid and polyploid. On the contrary, signet ring cells usually belong to diploid. It seemed logical that monocytoid carcinoma cells would have the greatest value in the early diagnosis of this carcinoma.


[The expression and significance of blood group antigens (BGA) A, B, H, Le(a) and Le(b) in hepatocellular carcinoma and chronic hepatitis]. Five monoclonal antibodies which selectively recognize A, B, H, Le(a) and Le(b) BGA had been used for immunohistochemical examination of serial sections collected from 40 cases of hepatocellular carcinomas, 63 cases of chronic hepatitis, 10 cases each of fetal and normal adult liver. All of the cases had been followed-up except the autopsy cases. The results were as follows: in fetal and normal adult liver tissues, none of the liver BGA were detected in the parenchymal liver cells. Five BGA were expressed in 11 hepatitic cases (17%) and also expressed in 19 hepatocellular carcinomas (48%). Combining the expression of BGA with the follow-up results, the study we have performed indicates that the expression of BGA in chronic hepatitis predicts cancerous change or severe liver cell damage associated with poor prognosis.


[The expression of nuclear estrogen receptor and its relation to cytoplasmic estrogen receptor in breast cancer]. Sixty specimens of breast cancer were assayed with ER-monoclonal antibody by immunocytochemical staining. Twenty-nine (48.33%) were nuclear estrogen receptor positive (ERn+). The number of ERn+ cancer cells decreased in the following sequence: mucinous carcinoma, invasive lobular carcinoma, invasive ductal carcinoma, and papillary adenocarcinoma. Two apocrine carcinomas were ERn-. ERn+ rate was higher in patients over 60 years of age (P less than 0.05). The amount of ERn+ cells was much greater in cancer cells than in the surrounding benign epithelial cells. This phenomenon may indicate that malignant cells are more hormone-dependent than benign cells. The results of immunocytochemical staining and steroid binding assay were compared. By immunocytochemical staining, twenty-four of fifty-seven cases (42.11%) were ERn+ and ERc+. Sixteen cases (28.07%) were ERn- and ERc-. This study showed that in a number of breast cancers ERs were positive in both cytoplasms and nuclei and the concordant rate was 70.18%. In the remaining cases 13 (22.81%), ERs were positive in the cytoplasm, and in 4 cases (7.02%) positive in nuclei only. Additionally, fifty-two out of 60 cases were assayed by 3H-estradiol and 3H-R5020 by means of steroid binding assay. Twenty-seven cases of them (51.92%) showed ERc+ and PRc+, and seventeen cases (32.69%) were ERc- and PRc-. Their correspondent rate was 84.61%.


[Flow cytometric analysis of colorectal cancer and polyps]. Thirty-four cases of colorectal cancer, 24 cases of polyps of various types and 30 cases of control colorectal mucosa were analysed by flow cytometry for DNA content in order to study their relation to histopathology and prognosis. The result showed that the DNA content of 6 out of 24 polyps (25%) were aneuploid, and all of these 6 cases with aneuploid DNA content were adenomas accompanied with mild to severe dysplasia, and six cases of juvenile polyps were diploid. There was a correlation between DNA aneuploid and severe dysplasia (P less than 0.05), but not with the histopathologic type. Twenty-three out of 34 cancers were aneuploid (68%), and correlation was noticed between the DNA Index (DI) and Dukes' classification (F = 17.37), but not with the differentiation of tumors. Patients with DNA aneuploid tumours had a worse prognosis than those with only DNA diploid tumours. Therefore, DNA ploidy of colorectal cancers detected by flow cytometry is of prognostic value for colorectal cancer.


[Activation of delta-opiate receptor simultaneously stimulates ACTH beta-endorphin and prolactin release]. To evaluate a possible physiological and pathological role of brain delta-opiate receptor and endogenous ligands in the control of ACTH, beta-endorphin (beta-EP) and prolactin (PRL) release, a specific agonist of the delta-opiate receptor--[D-Pen2, D-Pen5]-enkephalin (DPDPE) was microinjected into the third cerebral ventricle (1 microgram or 3 micrograms/3 microliters/rat) in conscious freely-moving male rats. Plasma ACTH, beta-EP and PRL concentrations were measured by radioimmunoassay (RIA). Our results clearly indicate that third ventricular injection of DPDPE significantly elevates plasma ACTH, beta-EP and PRL levels in comparison with control values, and that the stimulatory effects of DPDPE on the release of the three hormones are dose-related at 1 and 3 micrograms. The data suggest that activation of the delta-opiate receptor may be a mechanism behind the concomitant release of ACTH, beta-EP and PRL during stress and certain pathologic conditions.


[Effect of TRH on anti-restraint stress in rats]. Strong stress in response to restraint was observed in rats. In these rats, plasma ACTH increased significantly (P less than 0.001) while T3 and T4 decreased (P less than 0.05). After the rats were injected intraperitoneally with TRH, the plasma ACTH increase was obviously attenuated (P less than 0.001), while T3 INS and GLU increased (P less than 0.05) as compared with control rats. The results indicate that peripheral TRH participates in ACTH regulation during restraint stress.