Hemostatic changes in human adoptive immunotherapy with activated blood monocytes or derived macrophages. Human blood monocytes (Mo) and monocyte-derived macrophages (M psi) possess cytotoxic effects against tumor cell lines when appropriately stimulated by various biological response modifiers, e.g., gamma interferon (gamma IFN) and muramyltripeptide (MTP). Activated Mo/M psi represent a new tool for the treatment of human malignancies, termed "adoptive cellular immunotherapy". Activated Mo/M psi express tissue factor procoagulant activity (PCA), which is a physiological trigger of blood coagulation. PCA was evaluated in vitro using a modification of the one-stage recalcification clotting time, and hemostatic changes were studied in vivo in cancer patients. Nine patients with peritoneal carcinomatosis were injected intraperitoneally with activated Mo and 11 patients with non-small cell lung carcinomas were infused intravenously with activated M psi. Hemostatic changes were followed using activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen level, antithrombin III (ATIII) and protein C (PC) activities. Fibrinolytic activity was estimated by euglobulin lysis time and assays for plasminogen and fibrin/fibrinogen degradation products (FDP). These assays were performed before and after each autologous infusion and on days 2 and 3. Activated Mo and M psi expressed potent PCA (85.5 +/- 7.5 U/ml for MTP activated Mo and 50 +/- 5.3 U/ml for gamma IFN activated M psi suspensions). In both groups of patients, APTT, PT, and TT underwent no significant variations. There was no significant consumption of ATIII or PC, and fibrinolysis was not activated during the study period. In the group injected intraperitoneally with MTP-activated Mo, fibrinogen showed a significant and progressive increase in relation to the development of an inflammatory reaction, reaching a maximum average value of 6.1 g/l at the end of the therapy with a concomitant increase in FDP levels. This increase was not observed after intravenous therapy with gamma IFN-activated M psi. No patient suffered from hemorrhagic or thrombotic events. In our experience, repeated injections of activated Mo or M psi expressing potent tissue factor PCA did not induce significant in vivo activation of the coagulation system in cancer patients.


Filtration and local synthesis of lacrimal proteins in acquired immunodeficiency syndrome. In AIDS the onset of the ocular dry syndrome, characterized by lacrimal hyposecretion and deterioration of the corneal and conjunctival epithelium, generally accompanies the clinical aggravation of immunodepression. The study of serum and lacrimal proteins contributes to our knowledge of the pathophysiology of this syndrome. The lacrimal clearance of albumin indicates changes in the permeability of the haemato-lacrimal and conjunctival barrier. Lacrimal monomeric IgA and IgG are mainly of plasmatic origin, while polymeric IgA and IgM are synthesized in situ. The concentrations of these analytes thus reflect ocular humoral immune status. They show a strong humoral protein response in patients with cytomegalovirus retinitis. Lacrimal concentrations of lactoferrin and lysozyme were found to be significantly decreased in AIDS patients with ocular dryness, reflecting a decrease in the secretory activity of the lacrimal gland. Moreover, ocular lympho-plasmocyte infiltration was observed in several patients, with an increase in lacrimal beta 2-microglobulin concentrations. These various lacrimal proteins could be good markers of the ocular dry syndrome in AIDS.


Effects of beta-carboline on fear-related behavioral and neurohormonal responses in infant rhesus monkeys. We examined the effects of the inverse benzodiazepine agonist ethyl-beta-carboline-3-carboxylate (beta-CCE) on behavioral, hormonal, and neurochemical responses in infant rhesus monkeys exposed to fearful situations. Our paradigm elicits three distinct adaptive patterns of defensive behavior. From previous work, we hypothesized that behaviors induced by attachment bond disruption are predominantly mediated by opiate systems, whereas behaviors induced by the threat of attack are mediated by benzodiazepine systems. When beta-CCE (0, 125, 250, and 500 micrograms/kg) was administered immediately after maternal separation, the 500 micrograms/kg dose increased freezing and the 250 and 500 micrograms/kg doses reduced environmental exploration. Test conditions produced increased plasma ACTH and cortisol concentrations and increased cerebrospinal fluid (CSF) concentrations of MHPG and DOPAC; beta-CCE did not further affect these metabolites. A dose of 1000 micrograms/kg of beta-CCE increased CSF concentrations of DOPAC and MHPG in infants left with their mothers. During test conditions, it further increased CSF MHPG (but not DOPAC) concentrations, and reduced cooing while increasing freezing and barking and other hostile behaviors. Our results thus confirm that benzodiazepine systems mediate threat-related behaviors and suggest that coos, which were thought to predominantly reflect the degree of distress during separation, can be modulated by the infant's level of fear. beta-CCE also activated stress-related pituitary-adrenal hormonal systems and brain norepinephrine (NE) and dopamine (DA) systems. These effects occurred when animals remained undisturbed in their home cages with their mothers, suggesting that benzodiazepine receptors directly modulate brain NE and DA systems.


Gap junctions in myometrial cell cultures: evidence for modulation by cyclic adenosine 3':5'-monophosphate. Primary cultures of myometrial cells from juvenile rats, continuous cultures maintained by serial passage, and a pSV3neotransfected myometrial cell line were established and utilized for the study of development and modulation of gap junctional intercellular communication (GJIC) in vitro. The smooth muscle origin and homogeneity of the cultures were verified by immunofluorescence staining of alpha-smooth muscle actin and cellular desmin. Although gap junctions were not detected in thin sections of juvenile and adult myometrial tissues by transmission electron microscopy, they were detected in cultured myometrial cells derived from juvenile and adult animals. The presence of GJIC in cultured cells was confirmed using a fluorescence recovery after photo-bleaching assay. Administration of exogenous estradiol-17 beta (10(-7) M) resulted in an increase in GJIC in primary and passage 9 myometrial cultures, whereas pSV3neo-transfected myometrial cells were not significantly different from untreated controls. The lack of estrogen responsiveness in pSV3neo-transfected cultures correlated with lower levels of estrogen receptors than in primary cultures. Addition of 1 mM 8-bromo-cAMP resulted in rapid (within 2 min) increases in dye transfer in both control and estradiol-17 beta-primed primary cultures. Uncoupling of cells by treatment with 1 mM 1-octanol, followed by addition of 1 mM 8-bromo-cAMP, resulted in increased GJIC in control and estradiol-17 beta-primed cultures, although up-regulation of GJIC in estradiol-17 beta-primed cultures was much greater than in control cultures. Comparative experiments carried out on a spontaneously immortalized rat granulosa cell line (SIGC), which expresses the same connexin43 species as myometrial cells, exhibited similar responses to exogenous 8-bromo-cAMP following uncoupling of gap junctions with octanol. While the results of these investigations may not be extrapolated to myometrium in vivo, they suggest that myometrial cell culture may offer additional opportunities to explore the temporal expression and modulation of GJIC in myometrium.


Colorectal cancer genetics.

Colorectal cancer genetics. Due to the accessibility of the intermediate steps in the progression of colorectal cancer and to the existence of heritable susceptibility to the disease, molecular genetic analysis of colorectal carcinogenesis seems likely to answer many of the questions concerning the fundamental nature of the common human epithelial cancers. Several genetic events appear to be required and, although there is no stringent adherence to any particular sequence of events, the accumulation of genetic defects does show some loose order. Each event must confer growth advantage in order to allow further clonal expansion. Such expansion then makes further events at other crucial loci more likely. Hence, the process proceeds until the tumour is capable of the destructive growth, infiltration and metastasis characteristic of malignancy. This review summarises recent important progress in our understanding of both constitutional and somatic molecular genetic events involved in the development of colorectal cancer. Germline changes responsible for syndromes, such as Familial Adenomatous Polyposis, which result in predisposition to large bowel neoplasia are discussed. The possibility that heritable mutations in tumour suppressor genes might confer susceptibility to apparently sporadic colorectal cancer is proposed.


Peroxidase-negative and myelomonocytic antigen-positive acute leukemia. Between 1983-1988 bone marrow samples obtained from 195 peroxidase-negative leukemia patients were analyzed for their surface antigens. Thirteen of these patients (6.7%) had myelomonocytic-positive and lymphoid-negative antigens. These leukemic cells reacted with CD13 in eight patients, CD33 in seven, CD11 in six and CDw41 in two. In none of these patients did the leukemic cells react with CD1, CD2, CD3, CD4, CD5, CD8, CD10, CD19 or CD20. Leukemic cells from two patients were reactive with CD7. These leukemic cells demonstrated L2 morphology in 11 patients and L1 morphology in one patient. The leukemic cells from the final patient were diagnosed as those of leukemic transformation of myelodysplastic syndrome. Chromosomal abnormality was observed in approximately half of the patients examined (6/10). Cytochemical analysis revealed that the leukemic cells were negative for periodic acid Schiff stain but positive for acid phosphatase. The prognosis of these patients was markedly poor as compared to acute lymphocytic leukemia or typical peroxidase-positive nonlymphocytic leukemia. Complete remission was induced in only 30% of patients and duration of survival was short (4.7 months). This suggests that myelomonocytic antigen-positive peroxidase-negative acute leukemia is a distinct type of leukemia and may require more aggressive therapy to improve survival.


Herpes simplex virus type 1 and human immunodeficiency virus type 1 antigens in platelets from a hemophilia B patient with human immunodeficiency virus type 1-related thrombocytopenia. We analyzed platelet-associated antigens from a hemophilia B patient with human immunodeficiency virus type 1 (HIV-1)-related thrombocytopenia. Two bands appeared at 31,000 and 37,000 daltons in the platelet lysate after reaction with autologous serum in SDS-PAGE and Western blots. The band at 37,000 daltons was obtained using anti-herpes simplex type 1 (HSV-1) rabbit antiserum. Doublet bands at 36,000 and 37,000 daltons also appeared after reaction with HSV-1 seropositive human serum. The band at 31,000 daltons appeared after reaction with anti-HIV-1 rabbit serum. These results suggest that the platelet-associated antigens in this patient are components of both HSV-1 and HIV-1 antigens. In addition, acyclovir decreased his PAIgG level and increased his platelet count, and zidovudine increased his platelet count. Thus, we concluded that each of the platelet-associated antigens is partially responsible for the thrombocytopenia by causing deposition of immune complexes in this patient.


Human retroviruses.

Human retroviruses. It was only in 1980 that the first human retrovirus, HTLV-1, was isolated. Since then, HTLV-2, HIV-1 and HIV-2 have been identified. All four viruses are transmitted with varying efficiency sexually, vertically from mother to infant, and through blood by transfusion or contamination. HTLV-1 is endemic in populations in south-west Japan, Taiwan, sub-Saharan Africa, the Caribbean, southern USA, central and south America, Australia, Papua New Guinea, Solomon Islands and western Asia. There is now epidemic spread amongst IVDUs in north and south America and southern Europe. HTLV-1 is the aetiological agent of adult T-cell leukaemia/lymphoma (ATL) and tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). Other associations which may be causative are with polymyositis, infective dermatitis, gastrointestinal malignant lymphoma and chronic lymphatic leukaemia. ATL appears to be due to malignant transformation of HTLV-1 infected cells, and TSP/HAM to chronic activation of these cells. The epidemiology of HTLV-2 is being separated only recently from HTLV-1 through the application of PCR. It has a low level of endemicity in populations of central Africa, and central and south America. It is being spread epidemically amongst IVDUs in north America and southern Europe. Its association with any pathology in man remains uncertain. HIV-1 is epidemic and spreading rapidly throughout the world. In areas where homosexual contact was the predominant mode of transmission, heterosexual spread is becoming increasingly important. The areas where heterosexual contact is the predominant mode of transmission include the worst affected populations in the world, for example sub-Saharan Africa and some of the Caribbean. There have been recent and explosive increases of HIV-1 seroprevalence in IVDUs and female prostitutes in Asia, especially Thailand and India. Of the diverse pathology following infection, only the haematological consequences are reviewed in detail: these include anaemia, leucopenia, thrombocytopenia, disorders of coagulation and lymphomas. HIV-2, compared to HIV-1, is less infectious and causes less immunosuppression with more slowly progressive disease. It is prevalent in west Africa, but is spreading, albeit slowly, far beyond.


Studies on an outbreak of neonatal diarrhea caused by EPEC 0127:H6 with plasmid analysis restriction analysis and outer membrane protein determination. An outbreak of enteropathogenic Escherichia coli (EPEC) 0127:H6 diarrhea occurred at two nurseries for the newborn in Chongqing in May 1987. Sixty-nine neonates had diarrhea; two deaths resulted. The epidemic strains, carrying 1.5 and 60 Md plasmid DNA, had an identical restriction digest profile and the same outer membrane protein pattern and could produce localized adherence to HeLa, HEp-2 and FL cells. The rates of contamination with EPEC 0127:H6 on medical staff's hands in these two nurseries were 11.8% and 8.7%, respectively, whereas 85 samples from milk, air and other sources were all negative for EPEC. The source of infection was the index case's mother who had had watery stools. Transmission of EPEC 0127:H6 from infant to infant took place by way of the fecal-oral route, most likely via the hands of medical staff attending their care. We present the first case, confirmed by plasmid and restriction analyses and outer membrane protein determination, of a neonate who acquired EPEC during delivery through ingestion of organisms residing in the maternal birth canal.


Thiazide therapy for ACTH-induced hypercalciuria and nephrolithiasis. A one-year-old boy presented with hypercalciuria and nephrolithiasis following a course of ACTH therapy for infantile spasms. After a successful cystolithotripsy, therapy with chlorothiazide was followed by regression of the hypercalciuria within 42 months. Neither nephrolithiasis nor nephrocalcinosis recurred. Therapy with thiazides to prevent hypercalciuria caused by ACTH is proposed.


Managing service change in post HIV drugs services--a case study from Inner London. The advent of HIV/AIDS has presented a major ideological challenge to established drug treatment agencies, where the incorporation of HIV-prevention strategies has proved problematic. This case study analyses the response to the HIV/AIDS epidemic by the drug misuse services of one Inner London district health authority (Bloomsbury) during the period 1985-89. While innovation and enthusiasm were much in evidence, a number of special problems prevented an effective and coherent strategic response. Key factors in the management of change in drugs/HIV services are identified which may be of special importance to clinicians, managers and funders and a number of key organizational and managerial competencies are suggested.


The effect of a 'recall-system' in the treatment of alcoholic patients. In a service specializing in alcohol and drug abuse in Brazil, a 'recall system' was developed in order to decrease the drop out level, which is high in this kind of treatment. The effect of this procedure was measured in two groups: in the 82 patients with the 'recall system', the drop out rate after 12 months was 45%. In 227 patients without this system the drop out rate was 84%. The difference in drop out rate was significant (p less than 0.01) only during the first 6 months of treatment.


Development of cat somatosensory cortex: structural and metabolic considerations. Although research is beginning to clarify the relationship between structure and functional activity in the adult cerebral cortex, little is known about cortical development in the somatosensory cortex of cats. A number of parameters were used in this study to identify functional and anatomical correlates in the developing somatosensory cortex of kittens ranging in age from 3 to 33 d: 2-deoxyglucose (2DG) uptake, cytochrome oxidase (CO) activity, Nissl staining, and AChE activity. All of these parameters were found to reflect an immaturity that evolved to the adult-like pattern by 4-5 weeks of age. Nissl staining revealed an immature laminar pattern at birth, in which layers I, V, and VI were distinct whereas layers II-IV were homogeneous in appearance. Numerous cells could be observed at the layer VI-white matter junction and throughout the white matter, a feature not found in adults. The laminar distribution of Nissl-stained cells gradually became mature by 4-5 weeks of age. CO staining was homogeneous throughout all layers, in contrast to the adult pattern, which displays laminar differentiation. In young animals, many darkly stained CO+ cells were found at the layer VI-white matter border and in the white matter, a distribution not found in the adult. AChE staining in kittens was also distinctly different from that in adults. At birth, AChE+ fibers could be found in layers I, V, and VI but were scarce in layers II-IV. In the adult, a dense network of AChE+ fibers can be found in all layers. 2DG uptake was also immature, as little stimulus-evoked activity could be observed in animals younger than 2 weeks. A dense band of metabolic activity was found in the zone between layer VI and the white matter, whether or not a somatic stimulus was delivered. These results suggest a close correlation between the developing cytoarchitecture and the emergence of a mature pattern of functional activity in the somatosensory cortex.


Relationship between 1H and 13C NMR chemical shifts and the secondary and tertiary structure of a zinc finger peptide. Essentially complete assignments have been obtained for the 1H and protonated 13C NMR spectra of the zinc finger peptide Xfin-31 in the presence and absence of zinc. The patterns observed for the 1H and 13C chemical shifts of the peptide in the presence of zinc, relative to the shifts in the absence of zinc, reflect the zinc-mediated folding of the unstructured peptide into a compact globular structure with distinct elements of secondary structure. Chemical shifts calculated from the 3D solution structure of the peptide in the presence of zinc and the observed shifts agree to within ca. 0.2 and 0.6 ppm for the backbone C alpha H and NH protons, respectively. In addition, homologous zinc finger proteins exhibit similar correlations between their 1H chemical shifts and secondary structure.


Support of 1H NMR assignments in proteins by biosynthetically directed fractional 13C-labeling. Biosynthetically directed fractional incorporation of 13C into proteins results in nonrandom 13C-labeling patterns that can be investigated by analysis of the 13C-13C scalar coupling fine structures in heteronuclear 13C-1H or homonuclear 13C-13C correlation experiments. Previously this approach was used for obtaining stereospecific 1H and 13C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the 1H spin-system identifications. Studies of the hydrolysates of fractionally 13C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of 13C-13C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear 1H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the gamma- and delta-methyl groups of Ile. In addition to the methyl resonances, the gamma CH2 groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the delta-carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.


A triple-resonance pulse scheme for selectively correlating amide 1HN and 15N nuclei with the 1H alpha proton of the preceding residue. A 3D 1H-15N-13C triple resonance experiment is presented that contains exclusively cross peaks between the 1HN and 15N nuclei of one residue with the H alpha of the preceding residue. The pulse sequence, designed to minimize the time coherence, is transverse on nuclei with short T2 values. The experiment consists of coherence transfers via one-bond couplings from the HN via N, CO, C alpha to the H alpha and back to the HN for detection; it is called HN(COCA)HA. The experiment was tested on uniformly 15N- and 13C-enriched T4 lysozyme.


Dual infection with Pneumocystis carinii and respiratory viruses complicating bone marrow transplantation. Pneumonia due to dual infection with Pneumocystis carinii and respiratory viruses is a rare but formidable complication of bone marrow transplantation. We report here two cases of viral infections complicating P. carinii pneumonia in bone marrow transplant recipients who, at the time of infection, were not taking P. carinii prophylaxis. Both patients died following the pneumonia. Potential factors contributing to the dual infection included graft-versus-host disease, high-dose steroids and cyclosporin A. P. carinii prophylaxis should be continued for 12 months, or longer in bone marrow transplant recipients requiring prolonged immunosuppressive therapy. As specific antiviral therapy becomes available for some respiratory viral infections, performing regular viral surveillance cultures and responding with active early treatment may help improve the outcome in these immunocompromised patients.


Time-related decreases in mu and delta opioid receptors in the superficial dorsal horn of the rat spinal cord following a large unilateral dorsal rhizotomy. The aim of the present study was to measure the time-related modifications of mu and delta opioid binding sites in the superficial layers of the dorsal horn of the rat spinal cord after a C4-T2 unilateral dorsal rhizotomy. Using specific ligands, namely [3H]DAMGO for mu sites and [3H]DTLET for delta sites, and a quantitative autoradiographic analysis, we have observed: (a) a decrease in binding on the ipsilateral side to the lesion as early as the first day postrhizotomy, the maximal loss being attained at 8 days postlesion, (b) after 8 days postlesion, the residual binding remains stable over the period of analysis (90 days), (c) the loss of mu receptors (71-74%) is significantly more pronounced than the loss of delta receptors (57-62%) and (d) affinities of postsynaptic mu and delta receptors are similar to those of the total receptor population in the superficial layers of the dorsal horn. Comparison of these results with the degeneration of primary afferent fibers reported in literature favors the localization of the majority of mu and delta opioid binding sites on fine diameter primary afferent fibers.


Ultrastructural, biochemical and electrophysiological changes induced by 5,6-dihydroxytryptamine in the CNS of the snail Helix pomatia L. The serotonin neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT), was injected into the body cavity of snails. Changes induced in the central nervous system (CNS) by the neurotoxin were studied by morphological, electrophysiological and biochemical techniques for up to 90 days following injection. The neurotoxin induced a variety of ultrastructural alterations during the early phase (1st to 6th days) following treatment. On day 6 after treatment, membranous structures first appeared in the synaptic-like areas and apparently migrated to cell bodies where they were detected by day 14. Their number increased with time. Neurotoxin-induced structural alterations were found in neuronal processes and cell bodies of the serotonergic metacerebral giant cells injected intracellularly with horseradish peroxidase and in serotonin immunoreactive axons. These findings suggest that the toxin-induced alterations are rather selective for the serotonin-containing neuronal elements. The neurotoxin decreased the concentration of 5-HT in and [3H]5-HT uptake into cerebral and pedal ganglia, with a maximum effect between the 3rd and 5th day following drug administration. 5-HT levels and 5-HT uptake returned to normal by 19-21 days after treatment. The concentration of dopamine and of [3H]DA uptake capacity were reduced between 3-5 days after injection of 5,6-DHT by 6-7 days following treatment. The transmission from identified serotonergic synapses to targets was reduced beyond day 5 after 5,6-DHT administration. By 15 days after treatment, synaptic transmission between the metacerebral giant cell (MGC) and buccal followers was blocked. Transmission recovered by day 21 after 5,6-DHT. Comparison of the time-course of functional and structural recovery indicates that while functional recovery takes place within 21 days after treatment, certain structural alterations, e.g. the membranous structures and dense particles, remain in the nerve fibres and cell bodies. These may serve as specific intracellular markers of the serotonin-containing neuronal elements long after functional recovery from the effect of 5,6-DHT.


Corticosterone implants in the paraventricular nucleus inhibit ACTH and corticosterone responses and the release of corticotropin-releasing factor following neural stimuli. Experiments were conducted on the possible role of corticosterone (CS), at the level of the paraventricular nucleus of the hypothalamus (PVN) and dorsal hippocampus (D.HIPP), in the negative feedback effects following neural stimuli. In rats with bilateral PVN cholesterol (CHO) implants, acoustic and photic stimuli caused a significant rise in serum CS and ACTH and depletion of median eminence (ME) CRF-41 content. CS PVN implants have prevented the rise in serum CS, ACTH and ME CRF-41 depletion. Bilateral CHO or CS implants in the D.HIPP did not modify the responses of the hypothalamo-pituitary-adrenal axis to the above neural stimuli. In PVN CS-implanted rats, i.v. injection of CRF-41 increased serum CS similar to that observed in PVN CHO-implanted animals. These data are discussed in view of previous experiments on corticosteroid implants in the brain. It is concluded that CS PVN implants, by acting possibly via type II hypothalamic receptors, have prevented the release of ME CRF-41 following neural stimuli and consequently the secretion of ACTH and CS.


Gastric motility in conscious rats given oxytocin and an oxytocin antagonist centrally. Previous experiments have shown that gastric motility is inhibited by microinjection of oxytocin (OT) into the dorsal motor nucleus of the vagus (DMN) in anesthetized rats, and that the inhibition of gastric motility after electrical stimulation of the hypothalamic paraventricular nucleus (PVN) is blocked by microinjection of an OT receptor antagonist directly into the DMN. As an extension of these observations, the present series of studies demonstrate that gastric motility in unanesthetized, freely moving rats was reduced both by intracerebroventricular (i.c.v.) administration of OT and by electrical stimulation of the PVN, and that both of these inhibitory effects were blocked by i.c.v. administration of an OT antagonist. Moreover, an OT antagonist administered i.c.v. alone caused an increase in baseline gastric motility. However, pretreatment with the same OT antagonist i.c.v. did not block the inhibitory effects of systemic LiCl or cholecystokinin on gastric motility in conscious rats. These results suggest that oxytocinergic neurons exert a tonic inhibitory effect on gastric motility in rats, but that the inhibitory effects of LiCl and cholecystokinin on gastric motility are not primarily mediated by parvocellular OT-containing neurons projecting from the PVN to the DMN.


Parity-associated alterations of medial preoptic opiate receptors in female rats. Preoptic area opiate receptor density was measured by quantitative autoradiography using [3H]naloxone in female rats during their first and second pregnancies and lactations and in a separate group of ovariectomized, nulliparous animals. Opiate receptor density in the medial preoptic area (MPOA) was elevated on day 12 of gestation in both primigravid and multigravid rats when compared with ovariectomized subjects. MPOA receptor density was reduced in primiparous mothers on day 5 of lactation relative to pregnancy. In contrast, receptor density in the MPOA did not decline in multiparous (second lactation) rats relative to pregnancy levels. Opiate receptor density was significantly higher on day 5 of lactation in multiparous than in primiparous mothers. No difference in receptor density was detected in the adjacent lateral preoptic area among the treatment groups. An examination of hormone titers revealed that basal prolactin levels were significantly higher in primigravid than multigravid rats, and that during lactation prolactin titers were negatively correlated with MPOA opiate receptor density in the primiparous mothers. The data demonstrate that multiple pregnancies and lactations result in changes of MPOA opiate receptor density and of circulating hormone levels. The findings are discussed in terms of the concurrent changes in neural opiate sensitivity associated with multiparity.


Cytochrome oxidase activity in vagal and glossopharyngeal visceral sensory neurons of the rat: effect of peripheral axotomy. Cytochrome oxidase (CO) activity, an endogenous metabolic marker, was examined in visceral sensory neurons of the rat nodose and petrosal ganglia by using enzyme histochemistry. In the normal nodose and petrosal ganglia, nerve cells showed various degrees of staining intensity. The population of darkly stained neurons in the nodose ganglion was higher than in the petrosal ganglion. Axotomy of the peripheral axons of these bipolar sensory neurons was used to study potential changes in ganglionic cellular metabolism associated with loss of afferent inputs and/or injury. Peripheral axotomy had a significant effect on CO activity in the nodose ganglion. By 3 days after axotomy, darkly stained neurons decreased in number and lightly stained neurons, which were not observed in the normal ganglion, appeared in the nodose ganglion. At 7 days after axotomy, the average population of these lightly stained neurons increased to 29% in the nodose ganglion. Subsequently, the population decreased so that at 14 days and 21 days, 19% and 7% respectively of neurons were stained lightly. Even at 28 days after axotomy, the lightly stained neurons were still observed. In the petrosal ganglion, no remarkable change was observed at any stage after axotomy. These results suggest that metabolic activity decreases in some nodose neurons after peripheral nerve section.


Quantal analysis of suppressing action of baclofen on mossy fiber synapses in guinea pig hippocampus. Baclofen, a selective agonist at the gamma-aminobutyric acidB (GABAB) receptor, has been considered to reduce the release of transmitter from nerve terminals by acting on presynaptic GABAB receptors, in addition to its postsynaptic action. The purpose of this study has been to re-examine quantitatively the action of baclofen in the hippocampus by a rigorous quantal analysis. (+/-)-Baclofen suppressed field potentials and intracellularly-recorded synaptic potentials induced in the subfield CA3 by mossy fiber stimulation in thin transverse sections of the guinea pig hippocampus. The amplitude distribution of excitatory postsynaptic potentials (EPSPs) induced monosynaptically by a granule cell could be described by the Pascal statistics. Suppression of the unitary EPSPs by baclofen (1 and 5 microM) was accompanied by decreases both in mean quantal content (m) and by mean quantal amplitude (q). The reduction in q was smaller than expected from a decrease in the input resistance of the postsynaptic neuron. It was suggested that the presynaptic and postsynaptic actions of baclofen contribute almost equally to suppression of the transmission at 1 microM, whereas the presynaptic action predominates at 5 microM.


Ultrastructural localization of inositol 1,4,5-trisphosphate 3-kinase in rat cerebellar cortex. Subcellular localization of inositol 1,4,5-trisphosphate 3-kinase in the rat cerebellar cortex was studied immunohistochemically using a monoclonal antibody. Electron microscopy revealed intense immunoreactivity in the dendritic spines of Purkinje cells forming synapses with the parallel fibers, climbing fibers and recurrent collaterals of Purkinje cell axons. The labelling was associated with the hypolemmal cisternae, surrounding matrix and plasmalemma including the postsynaptic densities. Weaker immunoreactivity was present in the dendritic spines of basket cells and in certain segments of Purkinje cell recurrent collaterals. The postsynaptic regions of the dendritic trunks of Purkinje and basket cells were negative. These results indicate that inositol 1,4,5-trisphosphate 3-kinase is distributed amongst the spines of various synaptic relations with different electrophysiological properties, and that axon terminals of certain cell types are another functional site for the enzyme.


Spinal mechanisms of delta 9-tetrahydrocannabinol-induced analgesia. Cannabinoids have been demonstrated to be effective antinociceptive agents when given intravenously. In order to determine whether spinal antinociception can be achieved while minimizing psychotomimetic properties, the pharmacological activity of delta 9-tetrahydrocannabinol (THC) was evaluated after intrathecal injection in male ICR mice. Although delta 9-THC produced potent antinociception, it also caused hypoactivity, hypothermia, and catalepsy. Intrathecal administration of delta 9-THC in mice which had their spinal cord transected at T12 also produced potent antinociception suggesting a spinal component to the antinociceptive effect. Biodispositional studies of [3H]delta 9-THC demonstrated that brain levels of the drug following intrathecal injection in spinally transected animals were not sufficient to produce the antinociceptive effect. These studies suggest the involvement of a spinal component in the antinociceptive action of the cannabinoids.


Quantitative light microscopic autoradiographic localization of alpha 2-adrenoceptors in the human brain. In the present work the anatomical distribution of alpha 2-adrenoceptors in the human central nervous system was studied in detail by quantitative autoradiography using the selective alpha 2 agonist [3H]bromoxidine ([3H]UK-14304) as a ligand. Only postmortem tissues from subjects free of neurological disorders were used in this study. Very high or high densities of alpha 2-adrenoceptors were found along layers I and III in non-visual neocortex, layers III and IVc of the visual cortex, CA1 field--stratum lacunosum-moleculare--and dentate gyrus--stratum granularis--at the hippocampal formation, nucleus arcuatus at the hypothalamus, locus ceruleus, nucleus dorsalis of vagus and at the stratum granularis of the cerebellar cortex. Relevant densities of alpha 2-adrenoceptors were also observed along the remaining layers of neocortex, nuclei centralis, medialis and corticalis at the amygdala, anterior thalamic group and rotundocellularis nuclei, paraventricular and ventromedial hypothalamic nuclei, substantia innominata, superior colliculus--stratum zonale--and lateral periaqueductal area at the midbrain, nucleus tractus solitarii and dorsal horn--substantia gelatinosa--of the spinal cord. [3H]Bromoxidine specific binding was very low or negligible in the remaining brain areas. Although a general parallelism between the distribution of these receptors could be observed for the rat and human brain, dramatic species differences in the level of alpha 2-receptors were found in several brain areas, such as thalamus, amygdala or cerebellar cortex. In general, the distribution of alpha 2-adrenoceptors in the human brain found here was parallel to that described for the noradrenergic presynaptic terminals in the mammalian central nervous system, lending some weight to the proposed predominant presynaptic localization of these receptors. The relevance of the anatomical distribution of alpha 2-adrenoceptors in the human brain for a better knowledge of the neurochemistry of neuropsychiatric disorders is discussed.


Opioid receptor changes in weaver mouse striatum. The dopaminergic projection from the substantia nigra to the patch and matrix compartments of the caudate-putamen undergoes a spontaneous, early postnatal degeneration in mice which carry the weaver gene. The projection to nucleus accumbens is relatively spared. Dopaminergic afferents have been shown to be important modulators of striatal opioid receptor expression. In the present study, opioid receptor localization in the caudate-putamen and nucleus accumbens of control and weaver mice was examined by quantitative autoradiography. Mu, delta and kappa opioid receptors were differentially distributed in nucleus accumbens and in patch and matrix compartments of the caudate-putamen. In animals which were homozygous for the weaver gene, the density of mu opioid receptors in both patch and matrix compartments was unchanged with respect to control mice. In contrast, the density of delta and kappa opioid receptors was significantly decreased in weaver caudate-putamen and nucleus accumbens. The significance of these results with respect to opioid receptor expression and Parkinson's disease is discussed.


Increased densities of binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 in rat brain following portacaval anastomosis. Using quantitative receptor radioautography, binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 were studied in rats 4 week after end-to-side portacaval anastomosis and in sham-operated controls. Portacaval anastomosis resulted in region-selective increases in density of [3H]PK 11195 binding sites in cerebellum, pons greater than thalamus, cerebral cortex greater than hippocampus greater than striatum. Possible mechanisms implicated in these changes include (i) the action of endogenous ligands for the mitochondrial benzodiazepine receptor such as octadecaneuropeptide and (ii) neurotoxic actions of ammonia. In view of the proposed role of these receptors as modulators of intermediary metabolism and neurosteroid biosynthesis, such changes could contribute to the neurochemical mechanisms responsible for portal-systemic encephalopathy.


A role for protein kinase C in long term potentiation of nicotinic transmission in the superior cervical ganglion of the rat. The superior cervical ganglion of rats was perfused with Ringer solution containing hexamethonium to produce a steady, partial, nicotinic block. The compound action potential (CAP) evoked by supramaximal single shock stimulation of the cervical sympathetic trunk (CST) was recorded from the internal carotid nerve. Bolus injection of the protein kinase C (PKC) activators 4 beta-phorbol-12,13-dibutyrate (PDBu) or 4 beta-phorbol-12,13-diacetate (PDAc) produced a marked, prolonged, dose-dependent potentiation of the CAP amplitude (e.g. 90% decay 2 h). A non-PKC activating phorbol ester (PE), 4 alpha-phorbol-12,13-didecanoate, produced no potentiation. The PE-induced potentiation was antagonized by the PKC inhibitor H-7. In addition, after 1 h exposure to PDBu (3 microM) and recovery from the potentiation (e.g. 2-4 h), a second exposure to PDBu or PDAc produced no potentiation. A 5 s 40 Hz supramaximal train to the CST produced a long lasting potentiation of the CAP (long-term potentiation, LTP) as described previously. However, a similar train did not evoke LTP after perfusion for 1 h with PDBu. The train-evoked LTP was depressed by the PKC inhibitor H-7 at a concentration which antagonized the PE-evoked potentiation. These data suggest that (i) PKC activation potentiates nicotinic transmission, and (ii) a component of the train-evoked LTP is mediated by PKC.


Afferent projections in the spinal accessory nerve to the facial motoneurons of the cat. Stimulation of the accessory nerve evoked polysynaptic excitatory postsynaptic potentials (EPSPs) in the facial nucleus (FN) neurons of anesthetized cats. From the experiments with severance of C1-C3 dorsal roots, it is suggested that accessory afferents enter the brainstem through the accessory nerve. It was also found that stimulation of the solitary tract nucleus produced exclusively monosynaptic EPSPs in the FN neurons and the afferent volleys are most likely to be relayed at the solitary tract nucleus.


Inhibition of GABA-gated chloride channel function by arachidonic acid. The effects of arachidonic acid and its metabolites on gamma-aminobutyric acid (GABAA) receptor function were determined in rat cerebral cortical synaptoneurosomes. Incubation of synaptoneurosomes with phospholipase A2 decreased muscimol-induced 36Cl- uptake. Arachidonic acid, the major unsaturated fatty acid released by phospholipase A2, also inhibited muscimol-induced 36Cl uptake. Similar inhibition was obtained with other unsaturated fatty acids (docosahexaenoic, oleic) but not with saturated fatty acids (stearic, palmitic). The effect of arachidonic acid on muscimol responses was inhibited by bovine serum albumin (BSA), and BSA enhanced muscimol responses directly, indicating the generation of endogenous arachidonic acid in the synaptoneurosome preparation. The generation of endogenous arachidonic acid was also indicated by the ability of 2 inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguaiaretic acid (NDGA), to inhibit muscimol-induced 36Cl uptake. We conclude that arachidonic acid probably has both direct and indirect actions on muscimol responses since both enzyme inhibitors inhibited muscimol responses but did not prevent the effect of exogenously added arachidonic acid. In additional experiments, arachidonic acid metabolites generated by cyclooxygenase, prostaglandins D2, E2 and F2 alpha, each decreased muscimol responses; prostaglandins F2 alpha was the most potent inhibitor. Since the unsaturated fatty acids and their metabolites are most susceptible to peroxidation, a generating system of superoxide radicals was tested on muscimol responses. A combination of xanthine and xanthine oxidase inhibited muscimol-induced 36Cl uptake in a concentration-dependent manner. We propose that the inhibition of GABAA neurotransmission by arachidonic acid and its metabolites can lead to increased neuronal excitability. This mechanism may play an important role in the development of neuronal damage following seizures or cerebral ischemia.


Heterogeneity in EC50 and nH of GABAA receptors on dorsal root ganglion neurons freshly isolated from adult rats. GABA activates a Cl- current through the GABAA receptor/ionophore complex that influences excitability of neurons. Studies using expression of cloned cDNAs coding for different GABAA receptor/ionophore subunits suggest that the EC50 and Hill coefficient for GABA are influenced by subunit composition. However, no direct evidence for such heterogeneity has been reported for vertebrate neurons. I have investigated the heterogeneity of EC50 and Hill coefficients (nH) of isolated dorsal root ganglion neurons using the whole-cell patch clamp technique. The EC50 for GABA varied from 26 to 107 microM among neurons. nH calculated from the logistic equation varied from 1.18 to 2.0. A negative correlation was found between the EC50 and nH (r = -0.81). Both nH and EC50 differed between some cells. However, in some instances, nH differed between cells while EC50 values were similar, and in other cells, EC50 values differed and nH was similar. In addition, when cells were categorized according to action potential shape, the EC50 and Hill coefficients differed among cell types in some instances and were similar in other instances. These findings demonstrate that different pharmacological profiles for GABA can be observed in adult mammalian neurons. Selective distribution of such pharmacological subtypes of GABAA receptors may contribute to control of neuronal excitability.


GABAA receptors in the amygdala: role in feeding in fasted and satiated rats. The purpose of this study was to clarify further the site of action in the amygdala as well as functional characteristics of feeding in response to two GABA receptor agonists. Guide cannulae for microinjection were implanted stereotaxically in the rat just above the central nucleus of the amygdala (CNA). Microinjections of 0.05, 0.25, 0.5 or 1.0 nmol muscimol, a GABAA-selective receptor agonist, produced a dose- and time-dependent decrease of food intake in both the satiated and fasted rat. The bilateral injection of muscimol into the amygdala was more effective than a unilateral injection during the first 2 h, although the overall effects were similar. Microinjection of 0.1 nmol bicuculline methiodide, a GABAA receptor antagonist, into the CNA significantly blocked this inhibitory effect of 0.05 and 0.5 nmol muscimol again in both the satiated and fasted rat. Doses of 0.05, 0.5, 5.0 and 10.0 nmol of the selective GABAB agonist, baclofen, injected into homologous sites in the CNA did not alter food intake. These findings support the viewpoint that the amygdala and its central nucleus comprise a pivotal region involved in the mechanisms underlying the control of feeding behavior. Further, it is envisaged that hypophagic or anorexic responses are induced through the activation of GABAA receptors by the presynaptic release of GABA from neurons which form a component of the anatomical system for hunger and satiety.


Morphometry of spinal motor neurons in amyotrophic lateral sclerosis with special reference to chromatolysis and intracytoplasmic inclusion bodies. Lewy body-like hyaline inclusions (LBI), Bunina bodies (BB) and central chromatolysis are characteristic neuropathological features of spinal motor neurons in amyotrophic lateral sclerosis (ALS). Using histometric methods, we studied the spinal motor neurons of 4 patients with sporadic ALS and 3 neurological control patients to determine the possible relationship between these neuropathological features and cytoplasmic, nuclear and nucleolar size. In an ALS patient with a very rapid clinical course, many neurons with LBI were observed. Enlargement of nucleolar size and the nucleolar/nuclear ratio of neurons with LBI or BB were the only histometric difference compared to those of normal-appearing neurons. In ALS patients with an average clinical course, the number of neurons with LBI was lower, the number of neurons with BB was higher, and the nuclear and nucleolar sizes were smaller than those of normal-appearing neurons, particularly in chromatolytic neurons without LBI or BB. These data raise the possibility that motor neurons with LBI may represent an early stage in the neurodegenerative process, when the rate of protein synthesis is elevated as evidenced by their larger nucleolar size. Thus, the causative agent(s) of ALS may initially stimulate protein synthesis while in turn leading to down regulation of certain luxury function genes, culminating in neuronal dysfunction and death.


Sympathetic preganglionic neurons projecting to the adrenal medulla and aorticorenal ganglion in the rabbit. The distribution of sympathetic preganglionic neurons (SPN) projecting to the adrenal medulla and the aorticorenal ganglion in the rabbit was studied using a dual retrograde transport technique. The B subunit of cholera toxin (CTB) was injected into the left adrenal medulla and wheatgerm agglutinin-apo-horseradish peroxidase-7 nm gold (WGA-apo-HRP-gold) was injected into the left aorticorenal ganglion. Retrogradely transported CTB was detected by immunohistochemistry, while gold particles were detected by silver intensification. SPN projecting to the adrenal medulla were observed in segments T2-L2 of the spinal cord in 5 rabbits, with the majority of cells within segments T6-T11 (79%). SPN projecting to the aorticorenal ganglion were seen in segments T2-L1 of the spinal cord in 5 rabbits, with the greatest number of the cells within T6-T11 (81%). Only a small number of doubly labelled cells (1%) were found in two rabbits. The results suggest that despite their similar segmental distribution SPN projecting to the adrenal medulla or the aorticorenal ganglion belong to separate populations and few, if any, individual SPN have axonal projections to both locations.


Evidence that local non-NMDA receptors contribute to functional deficits in contusive spinal cord injury. To investigate the role of non-N-methyl-D-aspartate (non-NMDA) types of excitatory amino acid (EAA) receptors in traumatic spinal cord injury, we administered 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX), a potent and specific antagonist of non-NMDA receptors, to rats with a standardized contusive spinal cord injury. Focal infusion of NBQX into the injury site significantly reduced long-term hindlimb functional deficits as well as decreasing the time required for the rats to establish a reflex bladder. The results suggest that non-NMDA receptors at or near the injury site are involved in producing a portion of the functional deficits that result from contusive spinal cord injury.


Efferent projections from the flocculus in the albino rat as revealed by an autoradiographic orthograde tracing method. The terminal sites of floccular efferent fibers were investigated in the albino rat by an autoradiographic orthograde method. The corticonuclear fibers terminated in the caudoventral part of the lateral cerebellar nucleus and in the caudoventral region of the lateral part of the posterior interpositus nucleus. A few fibers from the rostral flocculus terminated in the granular cell layer of the basolateral part of the nodulus and uvula as mossy fiber type terminals. The projection to the nodulus and uvula was confirmed, by an additional retrograde HRP study, to originate from scattered spindle-shaped cells in the floccular stalk. The corticovestibular fibers terminated massively in the subnucleus y. The fibers passing through the subnucleus y divided into two bundles; one bundle coursed rostrally to terminate in the lateral and ventral parts of the superior vestibular nucleus, while the other bundle passed through the lateral and then ventral parts of the lateral vestibular nucleus, supplying a few terminals to these regions, to terminate sparsely in the rostral to intermediate part of the medial vestibular nucleus and the rostroventral part of the spinal vestibular nucleus. Some fibers passing through the lateral vestibular nucleus coursed rostrally to terminate in the medial part of the superior vestibular nucleus. Sparse terminals derived from the rostral flocculus were found in the prepositus hypoglossal nucleus. No definitive differential efferent projections were demonstrated in the rat flocculus.


Environmental dietary factors in colorectal cancer. Some unresolved issues. Unresolved epidemiologic issues in large bowel cancer include reasons for changes in incidence rates, differences in epidemiology of cancer in the distal and proximal colon and rectal cancer, and the optimal fat and fiber levels for low rates. The authors examined questions of suitable procedures for the assessment of intake of specific key nutrients and mode of cooking. International data by site and nutritional factors were compared. Fat and fiber intake in the United States, Japan, and Finland related to differences in rates. Laboratory animal model studies indicated that type of fat affected carcinogenesis. Colon cancer rates in men in the United States increased slightly, but started to decline slightly, as in women. Dietary factors play a key role in causation and prevention of large bowel cancer, with a diet of 25% fat and 25 g fiber recommended. Comprehensive school health education should be used to modify the nutritional habits of children.


Primary prevention of colorectal cancer through dietary modification. Because colorectal cancer is the second leading cause of cancer death in the United States and many developed countries, its primary prevention is of extreme importance. Environmental and dietary factors are considered responsible for 85-90% of all cases. Epidemiologic, animal, and biochemical studies suggest that diets high in total calories and fat and low in various dietary fibers, vegetables, and micronutrients are associated with an increased incidence. Of these factors, calcium and wheat bran have been used most extensively in recent trials. It has been reported that 1.5-2.0 g/day of calcium significantly decreases DNA synthesizing cells of high-risk patients. However, chronic wheat bran supplementation appears to decrease both rectal mucosal DNA synthesis and polyp recurrence. Several clinical trials currently are underway to evaluate the diet-colon cancer link. The results of these studies will help to determine the importance of dietary intervention in the reduction of the colorectal cancer risk.


The relationship of human papillomaviruses to anorectal neoplasia. This review begins with an overview of the anatomy of the anal canal, which is followed by a discussion of human papillomavirus (HPV) infections in the anogenital tract. The organization of the HPV genome and the function of the encoded proteins is discussed in relation to the oncogenic potential of these viruses. Particularly stressed are interactions with known tumor suppressor genes. Then the interaction of HPV with the host cells and some growth factors is reviewed. An important consideration is the synergy between this virus and other known carcinogenic factors. These include smoking, immunologic status, and other factors. Finally, the pathologic features of anal warts and malignant lesions are summarized with respect to their histologic findings and expression of viral subtypes.


Inhibitory effects of dihydroagarofuran sesquiterpenes on Epstein-Barr virus activation. To search for possible antitumor promoters, we carried out a primary screening of thirty-seven dihydroagarofuran sesquiterpenes from Tripterygium wilfordii Hook fil. var. regelii Makino and Euonymus sieboldianus Blume, using their possible inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation which is induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Some of these sesquiterpenes, triptofordin F-2 (Takaishi et al., 1988), 1,2,6,8,15-pentaacetoxy-9-benzoyloxy-4-hydroxy-beta-dihydroagarofuran and triptogelin A-1 (Takaishi et al., 1990) were observed to significantly inhibit the EBV-EA activation at low doses. Based on the results, the structural requirements for the activity of these compounds were discussed [corrected].


Transcriptional patterns of growth factors and proto-oncogenes in human glioblastomas and normal glial cells. To document over-expression of proto-oncogenes in tumors, it is necessary to determine the level of expression in the progenitor normal tissue. These studies compare the levels of nuclear transcription of a series of growth-factor related genes and proto-oncogenes in human glioblastoma cell lines with those in three normal glial cell populations. The unusual finding was that levels in the three normal glial cell populations varied considerably for several genes and thus overexpression of a specific gene in a tumor cell when compared to just one normal glial cell population would not necessarily represent overexpression. In this study, we compared the level of 17 genes in 7 tumors to the highest level of each gene found in any of three normal glial cell populations. Over-expression of PDGF-B in 4/7 glioblastoma cell lines, EGFR in 1/7, neu in 1/7 IGF-2 in 1/7 and ros in 2/7 was observed. The variation observed in the normal glial cell populations emphasizes the possibility that the normal glial cell populations represent different glial cell lineages and/or stages of differentiation and that the tumors could have arisen from different normal glial cells. Matching lineages of normal and tumor cells, probably by monoclonal antibody reactions, may be required to accurately define over-expression.


Involvement of p53 mutation in the development of human salivary gland pleomorphic adenomas. We examined the status of the p53 mutation, a putative tumor suppressor gene, as well as the expressions of myc and mos oncogene products in human salivary gland pleomorphic adenoma cells in culture derived from four individuals using techniques which enabled selective and favourable growths of tumor cells. Culture techniques empolyed in this study consisted of type I collagen gel-coated dishes and serum-free medium as substrates and growth medium, respectively. Cells grown under above conditions were subjected to the analyses of p53, myc and mos expression. When analyzed by both immunocytochemical staining and immunoblot, mutant forms of p53 specifically detected by PAb240 were observed in three of 4 cases. However, none of the 4 cases expressed myc and mos oncogene products. These results may imply a role for p53 mutation in the development of human salivary gland pleomorphic adenomas.


Primitive neuroectodermal tumor: CT, MRI, and angiographic findings. Primitive neuroectodermal tumors (PNETs) are uncommon CNS neoplasms found usually in the first decade of life. This article presents a proven case of this lesion in a 14-month-old boy located deep in the left frontal lobe. This lesion was studied by CT, MRI, and cerebral angiography. The radiologic findings of this tumor were assessed and compared with those cases reported in the medical literature.


Elevation of free beta subunit of human choriogonadotropin and core beta fragment of human choriogonadotropin in the serum and urine of patients with malignant pancreatic and biliary disease. Human choriogonadotropin (hCG), its free beta subunit (beta hCG), and the core beta hCG fragment (c beta hCG) were measured by highly sensitive time-resolved immunofluorometric assays in the serum and urine of 29 patients with pancreatic cancer, 7 patients with biliary cancer, and 45 patients with benign pancreatic or biliary diseases. The results were compared with those of an age- and sex-matched reference population of nonpregnant women and men. Of the various forms of hCG assayed in serum, beta hCG showed the best diagnostic accuracy, and c beta hCG was the best marker in urine. Elevated serum concentrations of beta hCG were observed in 72% of the patients with pancreatic cancer, in 6 of 7 patients with biliary cancer, and in 9% of those with benign disorders. The serum concentrations of c beta hCG were elevated in 45%, 57%, and 2%, respectively, and those in urine in 55%, 71%, and 11%, respectively. The molar concentrations of c beta hCG in serum were mostly lower than those of beta hCG. Thus beta hCG secreted into serum appears to be the main source of c beta hCG in urine. Provided that they are measured by sufficiently sensitive and specific assays, beta hCG in serum and c beta hCG in urine appear to be useful markers for pancreatic and biliary cancer.


Induction by bufalin of differentiation of human leukemia cells HL60, U937, and ML1 toward macrophage/monocyte-like cells and its potent synergistic effect on the differentiation of human leukemia cells in combination with other inducers. We have recently demonstrated that bufalin is a new potent inducer of the differentiation of human myeloid leukemia cells. The present work was carried out to examine further the effect of bufalin on the growth and characteristics of human leukemia-derived cell lines U937, ML1, and HL60. At concentrations of 5-10 nM, bufalin decreased the growth of ML1 cells preferentially at the G2 phase and U937 cells at the S and G2 phases of the cell cycle. Bufalin, under these conditions, induced the differentiation of U937, ML1, and HL60 cells to monocyte/macrophage-like cells by measuring the expression of various differentiation markers, as assessed by morphology and histochemistry, and ability to phagocytose latex particles, to reduce nitroblue tetrazolium, and to develop Fc receptors. U937 and ML1 cells started to differentiate at 4 and 6 h, respectively, after treatment with 10 nM bufalin and showed maximum differentiation 72 h later. At present, a mechanism for the bufalin-mediated induction of the differentiation of these human leukemia cells remains to be determined. The combination of bufalin with all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, 4'-demethylepipodophyllotoxin ethylidene-beta-D-glucoside (VP16), or human gamma-interferon synergistically induced the differentiation of HL60 and U937 cells. A similar effect on ML1 cells was observed with the combination of bufalin with VP16 or human rTNF-alpha. These results suggest that bufalin in combination with VP16, all-trans retinoic acid, 1 alpha,25-dihydroxyvitamin D3, rTNF-alpha, or gamma-interferon may be very useful in the differentiation of human leukemia.


Analysis of T-cell receptor alpha/beta variability in lymphocytes infiltrating a melanoma metastasis. Multiple experimental and clinical studies have suggested that the immune system may, to some extent, control the development of melanomas. The presence of tumor-infiltrating lymphocytes could reflect an in situ immune reaction directed to the malignant cells. The characterization of T-cell receptor (TCR) expressed by tumor-infiltrating lymphocytes is one way to precisely analyze these local T-cell responses. In this study, we have assessed the TCR alpha/beta variability in tumor-infiltrating lymphocytes from a subcutaneous metastasis of a melanoma patient. Using the anchored-polymerase chain reaction 268 TCR alpha and 266 TCR beta chain transcripts have been cloned and sequenced. Their analysis shows that the T-cell infiltrate is extremely diverse, with no preferential TCR gene segment usage.


Cytogenetic characterization of B-cell lymphomas from severe combined immunodeficiency disease mice given injections of lymphocytes from Epstein-Barr virus-positive donors. We analyzed the karyotype of 27 B-cell lymphomas of human origin that developed in mice with severe combined immunodeficiency disease following the injection of peripheral blood leukocytes from Epstein-Barr virus-seropositive donors. Three tumors had clonal abnormalities detected with conventional techniques, 2 had trisomy 11, and 1 had a del(6)(q21q25). One other tumor had trisomy 11 detected with fluorescence in situ hybridization. Twelve tumors had a normal karyotype, 11 tumors had nonclonal abnormalities (which included trisomy 9 or 12 in 3 or 2 tumors, respectively), and one tumor had a karyotype of 92,XXXX(75%)/46,XX(25%) by conventional cytogenetic analysis. Trisomy for chromosomes, 9, 11, and 12 are recurring abnormalities that have been observed in lymphomas associated with an immunocompromised state. Clonal or nonclonal abnormalities were observed in 8 of 11 tumors derived from 3 donors whose peripheral lymphocytes induced a high incidence of tumors in mice with severe combined immunodeficiency disease compared with a clonal abnormality and 2 nonclonal abnormal cells in 2 of 5 tumors derived from 3 donors whose lymphocytes induced an intermediate to low incidence. These observations suggest an association between a higher incidence of karyotypically abnormal cells in lymphomas and the increased tumorigenic potential of the lymphocytes that induced these tumors.


Effect on cancer cells of plasmids that express antisense RNA of human papillomavirus type 18. Some human squamous cell carcinomas contain DNA of human papillomaviruses (HPV) and express RNA from the E6 and E7 genes. We have examined the effect of plasmids that express antisense RNA of these genes on the growth of the human cancer cell lines HeLa, C4-1, and 1483, which contain HPV type 18 DNA. As controls, the human cancer cell line 183 and the Vero line of monkey kidney cells were used, which do not contain HPV. Plasmids were introduced into the cells by electroporation; cells that contained HPV type 18 accepted the antisense-expressing plasmids at a lower frequency than the cells that lacked HPV. Cell lines were developed from HeLa cells that contained sense- or antisense-expressing plasmids, and lines that contained antisense-expressing plasmids showed slower growth, reduced ability to form colonies in soft agar, and increased serum requirements. The use of antisense HPV RNA might be a suitable approach to gene therapy of HPV-expressing human cancers.


K-ras activation in non-small cell lung cancer in the dog. To investigate the role of K-ras mutations in canine non-small cell lung cancer, we first determined the nucleotide sequence of the normal canine K-ras gene and then examined 21 canine lung tumors for activating K-ras mutations. Canine K-ras was analyzed by direct sequencing of polymerase chain reaction products generated with oligonucleotide primers derived from the human K-ras sequence. Four nucleotide differences were found between the canine and human K-ras sequence from position 5 to 211. The deduced amino acid sequence of the canine gene was identical to that of the human. Activated K-ras alleles were detected in 5 of the 21 canine lung tumors examined. The activating lesions were point mutations, predominantly in codon 12. Of the 14 adenocarcinomas examined, 2 (14%) had K-ras mutations. Two of 5 (40%) adenosquamous carcinomas and the only large cell carcinoma also contained activated alleles. The overall frequency of K-ras point mutation in non-small cell lung cancer (25%) is similar to that reported in human non-small cell lung cancer. We conclude that K-ras activation by point mutation is associated with, but not necessary for, non-small cell lung cancer development in the dog.


Elevated DT-diaphorase activity and messenger RNA content in human non-small cell lung carcinoma: relationship to the response of lung tumor xenografts to mitomycin Cł. The enzyme DT-diaphorase (DTD; NAD(P)H:quinone oxidoreductase, EC 1.6.99.2), is an obligate two electron reductase which catalyzes reduction of a broad range of substrates, including quinones. We report here variations in DTD concentrations among different classes of lung tumors known also to vary in their responsiveness to cytotoxic agents. Small cell lung carcinomas (SCLCs) and cell lines derived from them have the low DTD activities and mRNA content characteristic of normal human lung, whereas non-small cell lung carcinomas (NSCLCs) have greatly elevated levels. DTD activity was increased up to 80-fold in NSCLC tumors relative to normal lung and 20-35-fold in NSCLC relative to SCLC cell lines. Increased DTD activity appeared to be a function of the NSCLC phenotype rather than a result of derivation from a cell type rich in DTD, since all histological classes of NSCLC showed this phenotype. In addition, where transfection of SCLC cell lines with the v-Ha-ras protooncogene caused a transition to a NSCLC phenotype, DTD activity was also elevated. Neuroendocrine-positive cells (SCLC, carcinoids, and a few NSCLC lines) typically had far lower DTD activities than did cell lines which lacked neuroendocrine markers (most NSCLC cells and mesotheliomas). High DTD activity may be exploited in the design of drugs which undergo bioreductive activation by this enzyme. Consistent with this, xenografts derived from NSCLC cell lines with high DTD that were grown in athymic nude mice were more susceptible to the antitumor quinone, mitomycin C, than were xenografts derived from SCLC cells containing low DTD. These data provide a mechanistic basis for the rational design of more effective bioreductive antitumor agents for use against NSCLC.


Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung. Aberrations of the p53 gene in 115 surgical specimens of non-small cell carcinomas of the lung were examined by single-strand conformation polymorphism analysis of polymerase chain reaction products. Structural abnormalities of the p53 gene were observed in 60 tumors (52%), i.e., 8 of 14 large cell carcinomas, 24 of 58 adenocarcinomas, 25 of 37 squamous cell carcinomas, and 3 of 6 adenosquamous carcinomas. Direct sequencing of abnormal DNA fragments revealed 45 single-base substitutions, 9 deletions or insertion of a short nucleotide sequence, and 3 two-base substitutions in 57 tumors. In the other 3 tumors, loss of one of the p53 alleles was observed, with no mutation in the other allele. Allelic loss of the p53 gene was observed in 14 of 43 informative cases (33%), and in 11 of the 14 cases the remaining allele was mutated. The aberrations of the p53 gene were not limited to a particular histological type or clinical stage. Their high frequency suggests that they were involved in the genesis of non-small cell carcinomas of the lung. The mutation frequency (46%) of the p53 gene in tumors carrying mutated ras genes was essentially the same as the overall frequency in lung cancers, suggesting that accumulation of mutations in these two genes in a tumor is a random phenomenon.


Human hepatoma gangliosides: occurrence of a novel I-type glycolipid with NeuAc alpha 2-6Gal structure. Gangliosides with NeuAc alpha 2-6Gal structure have been studied in human hepatocellular carcinoma. The gangliosides were purified to homogeneity by a DEAE-Sephadex A-25 column chromatography and by repeated silica beads column chromatography. Three gangliosides containing NeuAc alpha 2-6Gal structure were isolated and were structurally characterized by using monoclonal antibodies, proton nuclear magnetic resonance, fast atom bombardment mass spectrometry, methylation analysis by gas chromatography-mass spectrometry, and exoglycosidase treatments. The first compound was identified as NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer. The structures of 2 other components were concluded to be as follows: [formula: see text] The first compound is a ganglioside that is characteristic of human meconium. The second compound has the same structure as a ganglioside recently found by us (Taki, T., Rokukawa, C., Kasama, T., Kon, K., Ando, S., Abe, T., and Handa, S., J. Biol. Chem., 267: 11811-11817, 1992) in meconium. The third compound is a novel type of ganglioside having blood group I-type structure as the core sequence. In addition to these gangliosides, 5 others were detected, and all except for GM3 were glycolipids with neolacto-series core structure. These results suggest that enzymes for the synthesis of neolacto type and NeuAc alpha 2-6Gal structure of glycolipids are activated in hepatoma.


Accumulation of p53 protein correlates with a poor prognosis in human lung cancer. Mutations in the gene coding for the p53 tumor suppressor protein are common in a variety of human cancers. To assess the role of a putative mutated p53 protein in human lung cancer, a monoclonal antibody recognizing it was used in an immunoperoxidase detection system. A total of 114 cases of Stage I and II adenocarcinomas and squamous cell carcinomas were studied. The staining pattern was always intranuclear and heterogeneous. When the median or mean survival time was compared between cases, p53 accumulation had a statistically significant negative prognostic value. This was supported by a Kaplan-Meier survival plot of p53 producers and nonproducers. In 7 of 24 Stage II cases that were negative for p53 in the primary tumor, metastatic regional lymph nodes were p53-positive. These latter cases had greatly reduced survival times. Thus, p53 accumulation in primary tumors (and regional lymph nodes) may identify a subgroup of lung cancer patients with a prognosis of more aggressive disease.


Occurrence of p53 gene deletions and human papilloma virus infection in human head and neck cancer. Little is known regarding the molecular genetic events in head and neck carcinoma. Epidemiological evidence suggests that both alcohol and tobacco use are related to the development of these neoplasms, and viral infections have also been postulated to play a role in some tumors. Loss of p53 tumor suppressor gene function has been found in many malignancies and can occur through either gene mutation or by interaction with the E6 protein of oncogenic human papilloma viruses (HPV). Because the mucosal surfaces of the head and neck are exposed to mutagens and HPVs, we studied DNA derived from 30 stage I-IV squamous cell carcinomas of the head and neck (9 primary tumors and 21 early passage cell lines) for p53 gene mutations as well as for the presence of oncogenic HPV DNA. Exons 2 through 11 of the p53 gene were examined using single strand conformation polymorphism analysis followed by direct genomic sequencing of all variants. HPV detection was done using polymerase chain reaction amplification with HPV E6 region type specific primers as well as L1 region degenerate ("consensus") primers; HPV type was determined by restriction fragment length polymorphism analysis of the amplified fragment as well as by Southern blotting of genomic DNA. Sixteen of 30 tumors (53%) had p53 mutations and oncogenic HPV DNA was detected in 3 of 30 (10%) tumors, none of which had p53 mutations. The p53 mutational spectrum observed was characterized by equal frequencies of transversions (6 of 16), transitions (5 of 16), and deletions (5 of 16). This distribution of mutations differs from the spectrum of p53 mutation reported in esophageal (P = 0.05) and lung (P = 0.02) cancers, two other tobacco associated neoplasms. A previously undescribed clustering of 3 mutations at codon 205 was also observed. A trend toward a shorter time to tumor recurrence after treatment was noted for those patients with tumors exhibiting p53 gene mutations, and no relationship between p53 mutations and tumor stage or node status was noted. Alteration in p53 gene function appears common in head and neck cancer, and the mutational spectrum observed may reflect the role of different mutagens or mutagenic processes than those responsible for the p53 mutations in lung and esophageal neoplasms.


Chlorination of cellulose with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in lithium chloride-N,N-dimethylacetamide. Microcrystalline cellulose was chlorinated with N-chlorosuccinimide-triphenylphosphine under homogeneous conditions in LiCl-N,N-dimethylacetamide. At the early stage of the reaction only replacement of the 6-hydroxyl groups with chlorine was observed, and 3-hydroxyl groups were replaced at a lower rate with Walden inversion. The effects of reaction conditions on the extent of chlorination were studied in detail. More than two equivalents of chlorination reagents per glucose residue were necessary to attain a high degree of substitution (ds) by chlorine, and the maximum ds attained was 1.86. Chlorinated disaccharides were found in the hydrolyzates of chlorodeoxycelluloses hydrolyzed under mild conditions, and their structures were studied by mass spectrometry.


Syntheses of D-myo-inositol 1,4,5-trisphosphate affinity ligands. A mixture of 2,3,6-tri-O-benzoyl-4,5-di-O-benzyl-D-myo-inositol and 1,3,6-tri-O-benzoyl-4,5-di-O-benzyl-D-myo-inositol, obtained during our synthesis of D-myo-inositol 1,4,5-trisphosphate [C.E. Ballou and W. Tegge, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 94-98], was separated after tetrahydropyranylation of the free hydroxyl group in each. 2,3,6-Tri-O-benzoyl-4,5-di-O-benzyl-1-O- (tetrahydro-2-pyranyl)-D-myo-inositol was debenzylated and the two free hydroxyl groups were phosphorylated by a dibenzyl phosphoramidite procedure. The tetrahydropyranyl group was then removed, and phosphorylation at position 1 with benzyl 3-(benzyloxycarbonylamino)propyl di-N-isopropylphosphoramidite, followed by oxidation and deprotection, provided 1-[3-aminopropoxy(hydroxy)phosphinyl]-D-myo-inositol 4,5-bisphosphate. This compound was coupled to activated agarose to prepare an affinity matrix for the isolation of D-myo-inositol 1,4,5-trisphosphate-binding proteins, and it was coupled to 4-azido-2-hydroxybenzoic acid to give a product that was labeled with 125I to prepare a photoactivable derivatizing reagent. The new derivatives retain significant biological activity as assessed by their ability to stimulate the release of stored Ca2+ from the endoplasmic reticulum of permeabilized rat basophilic leukemia cells.


Assessment of left ventricular dysfunction in acromegalic patients using radionuclide ventriculography parameters. The left ventricular function of 14 acromegalic patients was investigated using radionuclide ventriculography. After labeling the red blood cells with 750-1,000 MBq 99mTc-pertechnetate, ECG-triggered left anterior oblique images were recorded. Fourier analysis was then performed on the time-activity curve of the left ventricle. The ejection fraction (EF), peak ejection rate, time to peak ejection (TPE), time to end-systole (TES), peak filling rate (PFR), time to peak filling (TPF), 1/3 EF, 1/3 filling fraction (FF), TPE/T, TPF/T and TES/T values (T: time interval for one heart beat) were calculated for each patient. Five patients (35.7%) had clinical cardiovascular symptoms. A decreased EF was observed in 28.5% of the patients. In comparison to the control group, the EF (53.5 +/- 5.5 vs. 60.8 +/- 5% p less than 0.009), 1/3 EF (14.45 +/- 3 vs. 20 +/- 4%, p less than 0.001), 1/3 FF (28.5 +/- 10.6 vs. 41 +/- 11%, p less than 0.02), TPE (158 +/- 33 vs. 132 +/- 35 ms, p less than 0.01), TPE/T (20.2 +/- 5 vs. 16 +/- 3.7, p less than 0.01) and PFR (2.4 +/- 0.5 vs. 2.9 +/- 0.4 EDC/s, p less than 0.005) were significantly different. It was found that TPE was prolonged and the early ejection function was decreased. Diastolic dysfunction was found in 5 (35.7%) patients; 21.4% of the patients had decreased PFR values although they had no cardiac symptom, hypertension and/or cardiomegaly. Scintigraphic parameters did not correlate with the presence of hypertension, cardiomegaly or cardiovascular symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)


Demonstration of the formation of hydroxyl radicals in acute myocardial infarction in man using salicylate as probe. Dihydroxybenzoic acid (DHBA) derivatives of acetylsalicylic acid (ASA) are formed in vivo by the action of the hydroxyl radical (OH.). In order to evaluate the possible formation of OH(.) in acute myocardial infarction (AMI) in man, 9 consecutive patients with a first episode of AMI (8 males, 1 female, mean age 50.3 years), treated with rt-PA, and 8 healthy volunteers (7 males, 1 female, mean age 29.8 years) were studied. All subjects received 100 mg ASA p.o. daily; venous blood samples were taken 30 min after the first dose (time 0) and then at 3-, 6-, 12-, 24- and 48 h and 5 days. Serum was analyzed by HPLC and electrochemical detection for 2,3- and 2,5-DHBA contents. 2,3-DHBA was present in all subjects with AMI and undetectable in healthy volunteers at all time points studied. Serum levels of 2,5-DHBA did not show statistically significant differences between AMI patients and healthy volunteers. These data support the hypothesis that hydroxyl radicals are formed during AMI in man.


Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells. In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.


Differential molecular dynamics and transmembrane fluidity gradients in canine myocardial sarcolemma and sarcoplasmic reticulum. The molecular dynamics of highly purified preparations of canine myocardial sarcolemma (SL) and sarcoplasmic reticulum (SR) were quantified by electron spin resonance spectroscopy (ESR). Canine myocardial SL and SR have substantially different motional regimes in their membrane interiors as demonstrated by alterations in the relative peak height ratios, peak widths and peak splittings in ESR spectra of 16-doxylstearate incorporated into SL and SR. Quantification of the apparent order parameters (S) of 16-doxylstearate in SL and SR by analyses of ESR spectra demonstrated that the interior of the SL membrane was substantially more immobilized than the interior of the SR membrane (e.g. S = 0.168 +/- 0.002 for SL and S = 0.128 +/- 0.003 for SR). In contrast, only modest differences in membrane dynamics near the hydrophobic-hydrophilic interface were present in SL and SR as ascertained by ESR spectra of the probe 5-doxylstearate incorporated into these membranes. Myocardial sarcolemma contained heretofore unsuspected amounts of cholesterol (1.4 +/- 0.1 mumol cholesterol/mg protein) while sarcoplasmic reticulum contained only small amounts of cholesterol (0.17 +/- 0.06 mumol cholesterol/mg protein). Model systems employing binary mixtures of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol demonstrated that the observed alterations in molecular dynamics were due, in large part, to the differential cholesterol content in these two subcellular membrane compartments. Taken together, these results demonstrate that these two functionally distinct myocardial subcellular membranes have markedly disparate molecular dynamics and transmembrane fluidity gradients which may facilitate their performance of specific functional roles during excitation-contraction coupling in myocardium.


Developmental distribution of platelet-derived growth factor in the mouse central nervous system. Immunoblotting and immunohistochemical techniques have been used to characterize the developmental changes in the distribution and relative quantity of platelet-derived growth factor (PDGF), an important mitogen and growth regulator for glial (and possibly neuronal) cells. PDGF exists as a dimer of two chains, A and B, and antibodies which are relatively specific for one chain or the other can be used to localize PDGF isoforms during development. We have also studied the distribution of PDGF receptor beta subunit (PDGF-R beta)-like immunoreactivity using an antibody probe. All 3 isoforms of PDGF are found in neural structures during development, beginning at about the midpoint of embryogenesis. Immunoblotting studies confirm the presence of PDGF isoforms in brain during embryonic and postnatal development, with the distribution and relative abundance of each isoform appearing to be independently regulated. Similarly, immunoblotting studies have verified the relative abundance and specificity of PDGF receptor beta subunit. The immunohistochemical findings confirm and extend these biochemical observations. Each PDGF chain (A and B) has a discrete localization during nervous system development, and the immunohistochemical distribution of PDGF-R beta is distinct from each of the PDGF isoforms. PDGF A-chain (localized with an antibody to PDGF(AA) dimers) appears to be found in growth cones of developing neurons in mid-embryonic brain development. By 11.5 days post-conception (embryonic day 11.5, E11.5) to E12, PDGF isoforms are found in apparent neurons in the basal plate (future ventral horn) of spinal cord. PDGF-R beta-like immunoreactivity is localized to the boundary cap region of the developing spinal cord at the same age. Similarly, at E13.5, all 3 PDGF isoforms are found, to varying extents, within cells of the dorsal root ganglia and trigeminal ganglia. At the same developmental stage, PDGF receptor protein is most prevalent in the nerves accompanying these structures. By E15, both PDGF isoform and PDGF receptor beta subunit immunoreactivity have declined to near-background levels in the sensory ganglia, while in the spinal cord and developing forebrain, levels of all PDGF-related proteins remain high.


Autocrine motility factor and its receptor: role in cell locomotion and metastasis. The ability to locomote and migrate is fundamental to the acquisition of invasive and metastatic properties by tumor cells. Autocrine motility factor (AMF) is a 55 kD cytokine produced by various tumor cells which stimulates their in vitro motility and in vivo lung colonizing ability. AMF stimulates cell motility via a receptor-mediated signalling pathway. Signal transduction following binding of AMF to its receptor, a cell surface glycoprotein of 78 kD (gp78) homologous to p53, is mediated by a pertussis toxin sensitive G protein, inositol phosphate production and the phosphorylation of gp78. Cell surface gp78 is localized to the leading and trailing edges of motile cells but following cell permeabilization is found within an extended network of intracellular tubulovesicles. Gp78 tubulovesicles colocalize with microtubules and extension of the tubulovesicular network to the cell periphery is dependent on the presence of intact microtubules. Gp78 labeled vesicles can be induced to translocate between the cell center and periphery by altering intracellular pH as previously described for tubulovesicles labeled by fluid phase uptake. Anti-gp78 mAb added to viable motile cells is localized to large multivesicular bodies which, with time, relocate to the leading edge. Binding of AMF to its receptor induces signal transduction, similar to chemotactic stimulation of neutrophil mobility, as well as the internalization and transport of its receptor to the leading edge stimulating pseudopodial protrusion and cell motility.


Voltage-dependent L-type calcium channels in the development and plasticity of mouse barrel cortex. Entry of calcium ions into the neuron is a triggering signal for initiation of several processes which may lead to modification of synaptic connectivity. The developmental changes of voltage-dependent L-type calcium channel (VDLCC) were studied using [3H]PN 200 110 nifedipine displaceable binding in the barrel cortex of mice, a model structure for studying cortical plasticity. In vitro binding autoradiography was used to examine quantitatively the pattern of [3H]PN 200 110 binding to brains of animals aged from 3 to 70 days. The binding values in the somatosensory cortex rose two-fold in the period examined, reaching a plateau in the 4th postnatal week. The laminar pattern of binding changed during development, with the locus of heaviest labeling shifting from layer IV to II/III in the third postnatal week and thin bands of labeling developing in layers IV and VI. A very faint barrel-like pattern of labeling in the barrel field was observed. Neither this pattern nor the binding values were altered by unilateral neonatal removal of all vibrissal follicles. Saturation studies of binding to crude synaptosomal fractions of cerebral cortex of mice aged 3, 15, 28 and 70 days revealed the presence of a single binding site, with Bmax increasing from 48.7 +/- 5.1 fmol/mg protein at postnatal day 3 to 191.7 +/- 9.6 fmol/mg protein at day 70. No developmental changes in KD values were found. No correlation was found between the critical period for cytoarchitectonic plasticity of the barrels and the time when high values of VDLCC binding were observed.


Effects of methylcobalamin on diabetic neuropathy. We studied the clinical and neurophysiological effects of methylcobalamin on patients with diabetic neuropathy. In a double-blind study, the active group showed statistical improvement in the somatic and autonomic symptoms with regression of signs of diabetic neuropathy. Motor and sensory nerve conduction studies showed no statistical improvement after 4 months. The drug was easily tolerated by the patients and no side effects were encountered.


Positron emission tomography study of the human hypothalamus during normal ageing and in ischemic and degenerative disorders. Regional blood flow and oxygen metabolism were determined by positron emission tomography, using the steady state technique with 15O, in the hypothalamus and in the whole brain of fifty two normal persons and patients suffering from cerebral ischemia and degenerative dementia. During normal ageing regional blood flow and oxygen consumption appeared to increase slightly in the hypothalamus and to decrease in the whole brain in 24 persons. In the young age group the hypothalamus was more protected against ischemia than in the elderly group. In the aged group with cerebral ischemia and degenerative dementia regional blood flow and oxygen consumption were decreased in the hypothalamus to the same extent as in the whole brain.


Surgical management of the unruptured cerebral aneurysm accompanied by ischemic cerebrovascular disease. Unruptured cerebral aneurysms accompanied by ischemic CVD in 16 patients were analyzed and their managements are discussed. Unruptured aneurysms of 8 patients with mild or no clinical symptoms and small low density areas on CT were treated surgically. There was no operative mortality, but postoperative complication occurred in two patients. Both were operated on through the ipsilateral side of ischemic lesions shortly after the onset of ischemic symptoms. Eight patients who had severe neurological deficits due to ischemic lesions and large low density areas on CT did not receive aneurysmal operation. When unruptured aneurysms are accompanied by ischemic CVD, the treatment should be determined by the severity of neurological deficits caused by ischemic CVD. If an unruptured aneurysm was the source of emboli resulting in the ischemic lesion, it should be operated. Careful manipulation is required during surgery, especially from the ipsilateral side of ischemic lesion.


Bilateral traumatic extradural haematomas: report of 12 cases with a review of the literature. We are reporting 12 cases of traumatic bilateral extradural haematomas. The incidence was 4.8% of all cases of extradural haematomas. The most common site was the frontal region. CT scan examination was necessary for early diagnosis. Ten patients were operated upon while one patient was treated conservatively. Two patients died. The relevant literature is reviewed.


Ocular myopathy without ophthalmoplegia can be a form of mitochondrial myopathy. To characterize muscle pathology in 3 cases affected by ocular myopathy with eyelid ptosis and upper facial weakness, but without ophthalmoplegia, light microscopy and ultrastructural study were performed on levator palpebrae, orbicularis oculi and deltoid muscle biopsies. While levator palpebrae proved uninformative because of the massive fibrous degeneration of muscle, orbicularis oculi biopsies showed histochemical and ultrastructural alterations indicating a mitochondrial involvement, resembling that reported in ocular mitochondrial myopathies (OMM). On the other hand very mild aspecific findings were observed in deltoid. We suggest that these cases with ocular myopathy and without ophthalmoplegia should be considered a partial or initial form of OMM.


Relationships between dopamine infusions and intracranial hemodynamics in patients with raised intracranial pressure. Dopamine, 1-10 micrograms/kg body weight/min was infused in 6 patients suffering from cerebrovascular diseases with elevated intracranial pressure and a critical cerebral perfusion pressure. Dopamine decreased intracranial pressure in 3 and increased it moderately in the other 3 patients. In all patients, the dopamine-induced rise of mean arterial pressure led to an increase of cerebral perfusion pressure. Transcranial Doppler ultrasonographic recordings of the middle cerebral artery in patients whose intracranial pressure declined revealed a decrease of the pathologically elevated cerebrovascular resistance, and an augmentation of cerebral blood supply. In conclusion, dopamine infusions may improve cerebral hemodynamics in some patients with severe brain edema. Such patients can be identified by intracranial pressure and Doppler monitoring.


Muscle fiber conduction velocity in the diagnosis of sporadic hypokalemic periodic paralysis. A 6-year-old girl presented with episodes of profound muscle weakness since the age of 2 years. On the basis of decreased ictal serum potassium level and lack of metabolic disorder, primary hypokalemic periodic paralysis (HPP) was diagnosed. Both parents and 3 sibs were unaffected clinically. In all of them asymptomatic heterozygosity was very unlikely by the finding of normal muscle fiber conduction velocities, whereas in the patient interictal muscle fiber conduction velocity was lowered. Determination of muscle fiber conduction velocity can be helpful in documenting sporadic occurrence of HPP.


Non-invasive assessment of autonomic nervous function in Gilles de la Tourette syndrome. Autonomic nervous function was investigated in 18 Gilles de la Tourette (GTS) patients and in 23 controls, who matched the patients in age, sex, baseline blood pressure and baseline heart rate. Four heart rate tests were used (variation at rest, during deep breathing, following standing up and during a Valsalva manoeuvre), and two blood pressure tests (standing up and sustained handgrip). The only significant difference between the groups was found in the Valsalva test. This was due to the initial heart rate increase which was higher in the GTS group; the subsequent decrease did not differ between the groups. Increased sympathetic activity is a possible explanation for this finding, but no additional evidence in its favour was found. No signs of autonomic failure were found.


Giant axonal degeneration: determination of essential glycolytic enzymes. Aggregation and accumulation of intermediate filaments (IF) in different cell types is an ultrastructural hallmark of autosomal recessively inherited giant axonal degeneration (GAD) as well as of experimentally induced or occupationally acquired toxic polyneuropathies. Insufficient glycolysis caused by glycolytic enzyme deficiencies has been proposed as a likely cause of the IF accumulation. In this report data are presented on essential glycolytic enzymes in erythrocytes of a GAD patient and family. No significant changes were noted.


Infantile spasms in a patient with septo-optic dysplasia, partial agenesis of the corpus callosum and an interhemispheric cyst. An uncommon association of infantile spasms, septo-optic dysplasia, partial agenesis of the corpus callosum and an interhemispheric cyst is described in a child. The combination of these findings has not, to our knowledge, been previously reported.


Brain abscess after esophageal dilatation for stenosis. The case is presented of a 28-year-old female with a brain abscess after esophageal dilatation for stricture, secondary to an acute necrotizing esophagitis. Other causes of brain abscess were excluded. To our knowledge this is the first documented case of brain abscess after dilatation for esophageal stricture in adult life. Some reports in the pediatric literature have been published previously.


Kingella kingae, a rare cause of bacterial meningitis. A male adolescent with a history of pharyngitis developed meningitis due to Kingella kingae. This is a Gram-negative coccobacillus belonging to the family of Neisseriaceae. It is a rarely reported human pathogen, from which only 2 cases of meningitis have been described up to the present day. Our patient developed ophthalmoplegia, suggestive of basal meningitis. He was treated with penicillin G and recovered completely.


Fabry's disease presenting with stroke.

Fabry's disease presenting with stroke. An atypical patient with Fabry's disease is presented. This patient developed a left internal capsule lacunar stroke at the age of 25. The etiology of the stroke was unclear. At the age of 29 he was discovered to have corneal lesions suggestive of Fabry's disease but had no other clinical features typical of Fabry's disease. The diagnosis of Fabry's disease was confirmed biochemically. Fabry's disease should be included in the differential diagnosis of stroke of unclear etiology in young male patients.


Intraventricular meningioma of the fourth ventricle. A rare case of meningioma of the fourth ventricle is reported. A review of the literature revealed only 24 reported cases of this type of tumor.