Transforming growth factor-beta directs IgA switching in human B cells.

Transforming growth factor (TGF)-beta added to cultures of highly purified human splenic B cells induced high levels of IgA synthesis in the presence of PWM and activated cloned CD4+ T cells. TGF-beta had no effect on IgM or IgG production. The induction of IgA synthesis by TGF-beta reflected IgA switching, because a strong induction of IgA production was also observed, when sIgA- B cells were cocultured with cloned activated CD4+ T cells in the presence of pokeweed mitogen. Resting CD4+ T cell clones or activated CD8+, TCR-gamma delta + CD4-,CD8- T cell clones failed to provide the co-stimulatory signal that in addition to TGF-beta and pokeweed mitogen was required for induction of IgA switching and IgA synthesis. mAb against CD4 or class II MHC molecules inhibited TGF-beta induced IgA synthesis, indicating that CD4-class II MHC interactions are required for productive T-B cell contacts resulting in IgA production. In contrast, anti-LFA-1, anti-CD2, and anti-class I MHC mAb were ineffective. TGF-beta failed to induce IgA synthesis by sIgA+ B cells under these culture conditions. Interestingly, induction of IgA production by sIgA- B cells required neutralization of TGF-beta activity by addition of the anti-TGF-beta mAb 1D11.1G 24 h after onset of the cultures. IgA production was prevented when the anti-TGF-beta mAb was added at the start of the cultures, indicating the specificity of the reaction. IgA synthesis was completely suppressed when TGF-beta was present during the total culture period of 11 days. These findings indicate that TGF-beta can act as a specific switch factor for IgA, provided it is only present at early stages of the cultures.


Molecules involved in the adhesion and cytotoxicity of activated monocytes on endothelial cells.

Our study was designed to investigate the surface molecules involved in the adhesion and cytotoxicity of activated human monocytes on resting and IL-1-stimulated endothelial cells (EC). Monocytes, exposed to the prototypic activating stimuli IFN-gamma and LPS, showed increased binding to resting and IL-1-treated EC. Activated monocytes were cytotoxic for resting and IL-1-treated EC in a 24- to 48-h [3H]TdR release assay. Anti-CD18 mAb significantly inhibited binding of monocytes on EC: in particular they caused 59 and 22% inhibition of adhesion of activated monocytes to resting and IL-1-stimulated EC, respectively. Anti-VLA4 mAb had little or no effect on binding when used alone, but combined use with anti-CD18 revealed an important role for this adhesion pathway: in particular, VLA4-dependent adhesion accounted for 40% of the binding of activated monocytes on IL-1-treated EC. Anti-CD18 mAb caused similar inhibition (77 and 81%) of the cytotoxicity of activated monocytes on resting and IL-1-treated EC in spite of the fact that this pathway accounted for only 22% of binding to activated EC. Moreover, anti-VLA4 mAb, alone or in combination with anti-CD18, had no effect on cytotoxicity. These results suggest that adhesion of activated monocytes to activated EC involves the CD18- and VLA4-dependent pathways, but that the former is dominant for the expression of cytotoxicity. Thus, in the ensemble of adhesion molecules available for interaction between endothelium and activated monocytes, the hierarchy of their importance may vary for different functions.


Transforming growth factor-beta enhances the in vivo effector function and memory phenotype of antigen-specific T helper cells in experimental autoimmune encephalomyelitis.

Transforming growth factor-beta (TGF-beta) had a profound effect on the in vitro phenotypic development of Ag-activated Th cells and enhanced the in vivo effector function of these cells upon adoptive transfer. Previous studies have shown that there are two types of Th cell populations found in unimmunized animals, naive helper cells, which are short-lived and express low levels of CD44 and high levels of CD45R and Mel-14, and memory helper cells, which have a long life span and express high levels of CD44 and low levels of CD45R and Mel-14. Culturing of Ag-specific murine Th cell lines and clones in the presence of TGF-beta greatly enhanced both the memory phenotype of the cultured cells and the effector function upon adoptive transfer in experimental autoimmune encephalomyelitis. Histologic evaluation of spinal cords from recipients receiving passively transferred murine T cell lines cultured with TGF-beta revealed large demyelinated plaques (multiple sclerosis-like) that were not present in animals receiving cells cultured with Ag alone. TGF-beta also enhanced the capability of myelin basic protein-specific Lewis rat T cell lines to transfer experimental autoimmune encephalomyelitis and potentiated a purified protein derivative-specific rat helper cell line to transfer delayed type hypersensitivity. Thus, the effects of TGF-beta did not appear to be limited by species specificity, Ag specificity, or in vivo T cell function. This is the first study showing that TGF-beta can potentiate the development and maintenance of the Th cell memory phenotype in vitro and enhance their in vivo effector function in an animal disease model.


Neutrophil adherence to endothelium is enhanced via adenosine A1 receptors and inhibited via adenosine A2 receptors.

We have recently demonstrated that human neutrophils (PMN) possess two different classes of adenosine receptors (A1 and A2) that, when occupied, promote chemotaxis and inhibit the generation of reactive oxygen species (e.g., O2- and H2O2), respectively. We have previously demonstrated that adenosine protects endothelial cells (EC) from injury by stimulated neutrophils (PMN) both by diminishing generation of H2O2 and inhibiting adherence of PMN to EC. We therefore determined whether occupancy of A1 or A2 adenosine receptors regulated adherence of PMN to EC. At concentrations similar to those required to inhibit release of O2- by ligation of A2 receptors, both adenosine (IC50 = 56 nM) and 5'N-ethylcarboxamidoadenosine (NECA, IC50 = 8 nM), the most potent A2 agonist, inhibited adherence to EC by stimulated PMN (FMLP, 0.1 microM). In direct contrast, the specific A1 agonists N6-phenylisopropyladenosine and N6-cyclopentyladenosine (CPA) promoted PMN adherence to EC at concentrations of 1-100 nM. To further investigate the mechanisms by which adenosine receptor agonists affected the adherence of stimulated PMN we examined the effect of NECA (A2) and CPA (A1) on the adherence of PMN to fibrinogen (a ligand for the beta 2 integrin CD11b/CD18) and to gelatin. In a dose-dependent manner (IC50 = 2 nM), NECA inhibited the adherence of FMLP-treated PMN to fibrinogen- but not gelatin-coated plates. In contrast, CPA (A1) promoted adherence of stimulated PMN to gelatin-(EC50 = 13 pM) but not fibrinogen-coated plates. Theophylline (10 microM), an adenosine receptor antagonist, reversed the inhibition by NECA (0.3 microM) of stimulated neutrophil adherence to fibrinogen. These observations not only confirm the presence of A1 and A2 receptors on PMN but also suggest two opposing roles for adenosine in inflammation. Occupancy of A1 receptors promotes neutrophil adherence to endothelium and chemotaxis (a proinflammatory role) whereas occupancy of A2 receptors inhibits adherence and generation of toxic oxygen metabolites (an antiinflammatory role).


Zymosan-stimulated tumor necrosis factor-alpha production by human monocytes. Down-modulation by phorbol ester.

In this study, we showed that human monocytes produced TNF-alpha in response to zymosan, a particulate agonist. Protein kinase C (PKC) seems to play a regulatory role in zymosan-induced TNF-alpha secretion. The pretreatment of monocytes with PMA induced a dose-dependent inhibition of zymosan-stimulated TNF production. This inhibition was likely due to an activation of PKC because it was prevented by inhibitors of PKC, sphingosine, and staurosporine. Moreover, PMA elicited a profound down-modulation of zymosan binding to monocytes. The inhibition of zymosan binding and TNF production displayed similar dose-dependence, suggesting that both events were closely related. In addition, PMA did not modify the expression of CD11b/CD18 receptor that is involved in zymosan recognition. In view of these findings, qualitative changes of CD11b/CD18 molecules might account for the inhibition of zymosan binding and TNF production. Thus, PMA specifically increased the association of CD11b/CD18 with the detergent-insoluble cytoskeleton. Cytochalasin B but not microtubule disrupters, nocodazole and colchicine, partially prevented the inhibition of zymosan binding. Hence, the inhibitory action of PMA on zymosan binding seems to be mediated by an increase in attachment of zymosan receptor to cytoskeleton and more likely to microfilaments. The regulatory activity of PKC might represent a first way of limiting cytokine over-production in response to pathogens which interact with monocytes via CD11/CD18 molecules.


CD4+ Th2 response induced by Schistosoma mansoni eggs develops rapidly, through an early, transient, Th0-like stage.

Data from recent studies of murine schistosomiasis mansoni have indicated that certain characteristics of this infection, such as eosinophilia and elevated IgE, are due largely to the induction of Th2-like immune responses by parasite ova. The present study was designed to examine more closely the genesis and development of these skewed Th responses to schistosome eggs. Accordingly, eggs isolated from infected mice were injected s.c. into normal mice. After inoculation, draining lymph node (LN) cells were recovered, phenotyped, and tested for their ability to proliferate and secrete IL-2 and IFN-gamma (as markers of Th1 function) and IL-4, IL-5, and IL-10 (Th2 cytokines). The results show a maximal LN enlargement of 40- to 100-fold by day 3 after egg inoculation. The CD4/CD8/B cell ratio at this time is similar to that in LN from normal mice, but increases in numbers of cells expressing very low levels of MEL-14 and high levels of Pgp-1 are evident by days 3 and 10, respectively. Surprisingly, the initial detectable Ag-specific response to schistosome eggs, observed at day 1, is the production of IFN-gamma. By day 3, LN cells are capable of proliferating and making IFN-gamma plus IL-2, IL-4, IL-5, and IL-10 when stimulated with soluble egg Ag and, therefore, appear Th0-like. After 7 to 10 days, IFN-gamma production is severely depressed but the response continues to be characterized by IL-4, IL-5, IL-10, and IL-2. Depletion studies indicate that CD4 cells are the major population responsible for Ag-mediated proliferation and cytokine production. Results show that schistosome eggs are autonomous inducers of vigorous Th2-like effector responses. Further, our data, from a system that utilizes an in vivo priming step, support the contentions that skewed Th responses develop via an intermediate Th0 stage is accompanied by a loss of the MEL-14 surface marker and an increase in Pgp-1 expression.


Regulation of germ-line epsilon transcription and induction of epsilon switching in cloned EBV-transformed and malignant human B cell lines by cytokines and CD4+ T cells.

IL-4 induces germ-line epsilon transcript synthesis in normal human B cells, but a second signal provided by CD4+ T cells is required for subsequent production of productive epsilon mRNA and IgE synthesis. In the present study we demonstrate that IL-4 specifically induces germ-line epsilon transcripts in most EBV lymphoblastoid (LCL) and EBV+ and EBV- Burkitt's lymphoma (BL) cells without inducing IgE synthesis. However, cocultivation of cloned sIgM+, sIgE- EBV-LCL or BL cells with activated CD4+ T cell clones in the presence of IL-4 resulted in IgE production in 13 to 24% of the wells. Analysis of rearranged DNA in IgE-producing cloned BL cells indicated that epsilon switching occurred through a recombination deletion event. IgE production is a stable feature of the cloned switched EBV-LCL and BL cells because these cells continued to produce relatively high levels of IgE in the absence of IL-4 and CD4+ T cells. Induction of IgE synthesis was blocked by IFN-gamma, IFN-alpha, and transforming growth factor beta (TGF-beta), but only TGF-beta inhibited IL-4-induced germ-line epsilon mRNA expression. These results suggest that the inhibitory effects of TGF-beta are mediated via inhibition of germ-line epsilon expression, whereas IFN-alpha and IFN-gamma block other steps in the regulatory processes resulting in induction of IgE synthesis by established human B cell line cells. Our results indicate that the same set of signals that induce normal B cells to switch to IgE synthesis also induce high frequency epsilon switching in cloned established EBV transformed and malignant B cell lines including classical cell lines such as JY and BL-2.


Modulation of intercellular adhesion molecule-1 expression on human melanocytes and melanoma cells: evidence for a regulatory role of IL-6, IL-7, TNF beta, and UVB light.

Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interactions of leukocytes and parenchymal cells and thus plays an important role in immunologic and inflammatory reactions. The expression of ICAM-1 that is found on many different cells such as melanocytes and melanoma cells is induced by various cytokines, including interferon-gamma (IFN gamma), interleukin (IL)-1 and tumor necrosis factor alpha (TNF alpha). Because expression of ICAM-1 in melanoma was found to correlate with increased risk of metastasis, the regulation of ICAM-1 expression on human melanocytes and melanoma cells was investigated. Foreskin-derived melanocytes and melanoma cell lines (A375, G361) were incubated with different cytokines and ICAM-1 expression was evaluated by fluorescence-activated cell sorter. IFN gamma, IL-1, IL-7, TNF alpha, and TNF beta significantly upregulated ICAM-1 expression in a dose-dependent manner. Most interestingly, the cytokine IL-6, which does not influence adhesion-molecule expression on other cells, significantly upregulated melanocyte and melanoma cell ICAM-1 expression. This effect was dose dependent and could be blocked by an IL-6 antibody. Irradiation with ultraviolet (UVB) light did not influence constitutive ICAM-1 expression on melanoma cells and melanocytes, but suppressed cytokine-induced ICAM-1 expression when cells were harvested 16 h after irradiation. These findings were further confirmed by Northern blot analysis, showing a marked accumulation of ICAM-1 mRNA after cytokine treatment, which was reduced by irradiation with UVB light. However, when UVB-exposed melanoma cells were cultured for at least 48 h induction of ICAM-1 expression was observed. These data indicate that, similar to other cells, ICAM-1 expression on melanoma cells and melanocytes is regulated by cytokines and that UVB light affects ICAM-1 expression on melanocytic cells in a biphasic manner.


Expression of keratinocyte transglutamine mRNA revealed by in situ hybridization.

Plasma membrane-bound transglutaminase (TGm) catalyzes the formation of cornified envelopes (CE) in terminally differentiating keratinocytes. The recent cloning of cDNA encoding rabbit TGm allows detailed studies of its gene expression and regulation. In the present paper, we describe the localization of TGm mRNA in rabbit tissues, as well as in normal and psoriatic human skin, as assessed by in situ hybridization. Furthermore, we correlate TGm mRNA localization with the distribution of the TGm protein detected by immunohistochemistry with a specific monoclonal antibody. In rabbit epidermis, TGm mRNA was expressed in suprabasal cells. The TGm protein was detected in the upper stratum spinosum and stratum granulosum. In rabbit esophagus, TGm mRNA and protein were already expressed to a high level in the first suprabasal cell layer, and their expression decreased in the more differentiated cells. In normal human skin, a small amount of TGm mRNA, restricted to the stratum granulosum, was found, whereas psoriatic skin samples contained high amounts of TGm mRNA in the suprabasal layers with a decreasing gradient into the rete ridges, i.e., the involutions of the epidermis into the dermal compartment. The TGm protein was absent from the rete ridges and confined to several cell layers expressing high levels of mRNA. There was virtually no difference between uninvolved psoriatic and normal epidermis.


[Immunohistochemical study of proliferating cell nuclear antigen (PCNA) in gynecological tumors and their related lesions].

We have investigated whether monoclonal antibody (PC10) of proliferating cell nuclear antigen (PCNA) could be useful as a marker of proliferating cells within formalin-fixed, paraffin-embedded tissue sections of 140 gynecological tumors and their related lesions. There was a positive correlation (r = 0.76) between the labelling index for PCNA and that for Ki67. Immunohistochemical staining for PC10 was confined to the nucleus and showed a diffuse or granular pattern or a mixture of both. The distribution of PC10 staining in non-neoplastic tissues was localized to proliferating cell compartments. In malignant tissues, the localization of the distribution of PCNA-positive cells came to be lost and the proportion of positive cells varied from case to case as well as from field to field within the same tissue section. The cases in which more than 31% of cells were positive for PCNA were as follows: Cervical squamous dysplasia 2/3, squamous carcinoma in situ 2/5, microinvasive squamous carcinoma 2/2, invasive squamous carcinoma 9/13, adenocarcinoma in situ 4/4, microinvasive adenocarcinoma 3/3, invasive adenocarcinoma 6/7, endometrial adenocarcinoma 6/25, ovarian epithelial malignant tumors 11/17, sex cord stromal tumors 2/14, and germ cell tumors 3/22. It is concluded that immunohistochemical staining for PC10 may be useful as a marker for proliferating activity of the cells both in normal and tumor tissues rather than for malignancy.


Comparison of hantavirus isolates using a genus-reactive primer pair polymerase chain reaction.

RNA of more than 40 hantavirus isolates, originating from rodents and humans of widely separated geographical areas, was copied to cDNA using reverse transcriptase and amplified by polymerase chain reaction (PCR). A genus-reactive oligonucleotide primer pair, flanking a 365 bp region of the G2 glycoprotein gene, was chosen for genus-reactive PCR. DNA products were digested with 20 restriction endonucleases and cleavage patterns were analysed. For strains of known sequence, the restriction patterns observed were consistent with those predicted from sequence data, demonstrating that the amplified products originated from target virus RNA. Further analyses suggested that all amplified viruses could be easily typed into one of five restriction patterns using only five enzymes. The categories identified by restriction analysis of PCR-amplified cDNA corresponded with serogroups established by plaque-reduction neutralization tests. This method may greatly simplify the identification of new hantavirus isolates.


Cell-specific alternative RNA splicing of an FMRFamide gene transcript in the brain.

Individual neurons synthesize different peptide neurotransmitters and neuromodulators. In general, specificity is achieved by transcriptional regulation of neuropeptide-encoding genes. In Lymnaea, the FMRFamide and GDP/SDPFLRFamide neuropeptides are encoded by separate exons. Here we provide evidence that the two exons are part of the same gene and that in neurons expressing the gene the two exons are spliced onto a common upstream exon encoding a hydrophobic leader sequence. In addition, in situ hybridization data show that there is mutually exclusive cytoplasmic expression of each of the neuropeptide-encoding exons. Thus, differential neuropeptide synthesis is likely to be regulated by an alternative splicing mechanism. The cellular specificity of these splicing events is remarkable and suggests that cell-specific alternative splicing may be of major importance in establishing neuronal diversity in this system.


Beta-adrenergic receptors: astrocytic localization in the adult visual cortex and their relation to catecholamine axon terminals as revealed by electron microscopic immunocytochemistry.

It has long been recognized that noradrenaline, the most abundant catecholamine within the visual cortex, plays important roles in modulating the sensitivity of cortical neurons to visual stimuli. However, whether or not these noradrenaline effects are confined to a discrete synaptic specialization or mediated by diffuse modulation of a group of synapses has remained an issue open for debate. The aim of this study was to examine the cellular basis for noradrenaline action within the visual cortex of adult rats and cats. To this end, I used electron microscopic immunocytochemistry to examine the relationship between (1) catecholamine axon terminals and beta-adrenergic receptors (beta AR), which, together, may define the effective sphere of noradrenaline modulation; and then (2) these putative sites for catecholamine modulation and axospinous asymmetric junctions where excitatory neurotransmission is likely to dominate. Antibodies against beta AR were used at light and electron microscopic levels on the visual cortex of rat and cat. Rat visual cortex was also labeled simultaneously for beta AR and the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), to determine the ultrastructural relationships between catecholamine terminals and beta AR. Immunoperoxidase labeling revealed that beta AR404, a polyclonal antibody directed against the C-terminal tail of hamster lung beta AR (beta 2-type), recognized astrocytic processes predominantly. In contrast, beta AR248, a polyclonal antibody directed against the third cytoplasmic loop, recognized neuronal perikarya as observed in previous studies. Dual labeling for beta AR404 and TH revealed that catecholamine axon terminals that contained numerous vesicles formed direct contacts with astrocytic processes exhibiting beta AR404 immunoreactivity. However, some catecholamine axon terminals that lacked dense clusters of vesicles were positioned away from beta AR404-immunoreactive astrocytes. Frequently, beta AR-immunoreactive astrocytic processes surrounded asymmetric axospinous junctions while also contacting catecholamine axon terminals. These observations support the possibility that, through activation of astrocytic beta AR, noradrenaline modulates astrocytic uptake mechanism for excitatory amino acids, such as L-glutamate. Astrocytic beta AR might also define the effective sphere of catecholamine modulation through alterations in the morphology of distal astrocytic processes and the permeability of gap junctions formed between astrocytes.


Proctolin activates an inward current whose voltage dependence is modified by extracellular Ca2+.

The pentapeptide proctolin modulates the activity of the rhythmic pattern generators in the crustacean stomatogastric nervous system. Proctolin strongly excites the lateral pyloric and the inferior cardiac neurons of the stomatogastric ganglion (STG), causing them to fire extended high-frequency bursts of action potentials (Hooper and Marder, 1987; Nusbaum and Marder, 1989a,b). We now report that proctolin depolarizes these cells maximally at membrane potentials close to the threshold for action potential generation. In voltage clamp, proctolin evokes an inward current, carried at least partially by Na+, that shows strong outward rectification. Removal of extracellular Ca2+ markedly increases the amplitude of the proctolin-evoked current and linearizes its current-voltage curve. The properties of the proctolin current make it ideally suited to contribute to the activity-dependent modulation of the pyloric network of the STG.


Extinction of fear-potentiated startle: blockade by infusion of an NMDA antagonist into the amygdala.

Data derived from in vitro preparations indicate that NMDA receptors play a critical role in synaptic plasticity in the CNS. More recently, in vivo pharmacological manipulations have suggested that an NMDA-dependent process may be involved in specific forms of behavioral plasticity. All of the work thus far has focused on the possible role of NMDA receptors in the acquisition of responses. However, there are many examples in the behavioral literature of learning-induced changes that involve the reduction or elimination of a previously acquired response. Experimental extinction is a primary example of the elimination of a learned response. Experimental extinction is well described in the behavioral literature, but has not received the same attention in the neurobiological literature. As a result, the neural mechanisms that underlie this important form of learning are not at all understood. In the present experiments, the fear-potentiated startle paradigm was employed to begin to investigate neural mechanisms of extinction. The results show that infusion of the NMDA antagonist D,L-2-amino-5-phosphonovaleric acid (AP5) into the amygdala, a limbic structure known to be important for fear conditioning, dose-dependently blocked extinction of conditioned fear. Control experiments showed that the blockade of extinction was neither the result of the permanent disruption of amygdaloid function nor the result of decreased sensitivity of the animals to the conditioned stimulus. Infusion of AP5 into the interpositus nucleus of the cerebellum, a control site, did not block extinction. Finally, intra-amygdala infusion of a selected dose of the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione did not block extinction of conditioned fear. These results, together with a previous report from our laboratory (Miserendino et al., 1990), demonstrate the importance of the amygdala in the elaboration of conditioned fear and suggest that an NMDA-dependent process might underlie the extinction of conditioned fear.


Pharmacological discrimination of N-type from L-type calcium current and its selective modulation by transmitters.

GABA and norepinephrine inhibit high voltage-activated calcium current in chick sensory neurons. Using specific pharmacological tools, we have dissected this current into two components: the major one is omega-conotoxin sensitive and dihydropyridine resistant (N-type) while the minor one is dihydropyridine sensitively and omega-conotoxin resistant (L-type). The ability to selectively eliminate these two components has allowed us to determine whether the transmitters target the same or different channel types. Both GABA and norepinephrine modulate the N-type component as evidenced by their lack of effect on (1) omega-conotoxin-resistant current and (2) pure L-type tail current, prolonged by a dihydropyridine calcium channel agonist. This simple pharmacological profile will allow future tests of the significance of the two channel types in regulating sensory neuron functions.


Distribution and characterization of different molecular products of pro-somatostatin in the hypothalamus and posterior pituitary lobe of the Mongolian gerbil (Meriones unguiculatus).

Antisera raised against various synthetic peptide fragments of the pro-somatostatin molecule were used to visualize immunohistochemically the distributions of different pro-somatostatin fragments in the hypothalamus and posterior pituitary of the Mongolian gerbil. To define the nature of the immunoreactive somatostatin-related molecular forms, gel chromatography combined with radioimmunoassays of hypothalamic and posterior pituitary extracts was performed. Within the hypothalamus, only trace amounts of somatostatin-28 and somatostatin-28(1-12) were present, whereas pro-somatostatin(1-76), pro-somatostatin(1-64), and somatostatin-14 peptides were present in equimolar amounts. In contrast, the posterior pituitary lobe contained equal amounts of somatostatin-14, somatostatin-28, and somatostatin-28(1-12) but no pro-somatostatin(1-76), indicating that pro-somatostatin is further processed during the axonal flow to posterior pituitary nerve terminals. The gel chromatographic data were further substantiated by immunohistochemical data. Thus, perikarya containing all of these five immunoreactivities were strictly confined to the periventricular area and parvocellular subset of the paraventricular nucleus. However, the number of somatostatin-28- and somatostatin-28(1-12)-immunoreactive perikarya was approximately 20% of the number of somatostatin-14- and pro-somatostatin(1-64)-immunoreactive cells. In other hypothalamic areas only somatostatin-14 and pro-somatostatin(1-64) immunoreactivities were detectable in cell bodies. These cell bodies were encountered in the organum vasculosum laminae terminalis; the suprachiasmatic, ventromedial, arcuate, perifornical, and posterior hypothalamic nuclei; and the median preoptic and retrochiasmatic areas. In situ hybridization histochemistry revealed that the cellular distribution of pro-somatostatin mRNA corresponds to that of somatostatin-14 and pro-somatostatin immunoreactivity, suggesting that the immunoreactive material observed within the cell bodies is synthetized there and that the differences in density of immunoreactivities may be explained by intracellular processing of pro-somatostatin. Somatostatinergic nerve fibers and terminals in hypothalamic areas and the posterior pituitary lobe were immunoreactive to all of the employed antisera. From the present results, obvious differences between intrahypothalamic and hypothalamo-pituitary somatostatinergic neurons emerge. Within hypothalamic neurons not projecting to the median eminence and the posterior pituitary lobe, pro-somatostatin is posttranslationally processed in the cell body predominantly into pro-somatostatin(1-64) and somatostatin-14. Otherwise, within periventricular neurons projecting to the median eminence and the posterior pituitary lobe, pro-somatostatin is posttranslationally processed during the axonal flow into pro-somatostatin(1-64), somatostatin-14, somatostatin-28, and somatostatin-28(1-12).


Vasopressin modulates cerebrovascular responses to opioids in newborn pigs.

Vasopressin receptor blockade has been observed to attenuate the systemic vascular effects of dynorphin. This study was designed to determine the ability of vasopressin to modulate cerebrovascular responses to opioids in newborn pigs equipped with closed cranial windows. Topical dynorphin 13 increased pial arteriolar diameter during normotension (151 +/- 5, 171 +/- 4, 183 +/- 4 and 187 +/- 4 microns for control, 10(-10), 10(-8) and 10(-6) M dynorphin 13, respectively). During hypotension, however, responses to dynorphin 13 were reversed to concentration-dependent decreases in pial arteriolar diameter (184 +/- 3, 169 +/- 4, 165 +/- 4 and 159 +/- 4 microns for control 10(-10), 10(-8) and 10(-6) M dynorphin 13, respectively). Dynorphin 13-induced pial arteriolar dilation was potentiated by the V1 receptor antagonist [1-(beta-mercapto-beta beta-cyclopentamethylene propionic acid) 2(o-methyl)-Tyr-AVP] (MEAVP; 5 micrograms/kg i.v.; 14 +/- 1, 22 +/- 1 and 24 +/- 1% vs. 19 +/- 1, 26 +/- 1 and 30 +/- 1% increase for 10(-10), 10(-8) and 10(-6) M dynorphin 13 before and after MEAVP, respectively). In contrast, dynorphin 13-induced constriction during hypotension was markedly reduced by MEAVP (10 +/- 1, 15 +/- 1 and 16 +/- 2% vs. 1 +/- 1, 4 +/- 1 and 9 +/- 1% decrease for 10(-10), 10(-8) and 10(-6) dynorphin 13 before and after MEAVP, respectively). Dynorphin 8 and the synthetic kappa-opioid selective agonist, U5O,488H, elicited similar tone-dependent responses that were modified by MEAVP in a similar fashion.(ABSTRACT TRUNCATED AT 250 WORDS)


Cardiorespiratory effects of the novel opioid analgesic HP 736 in the anesthetized dog and conscious goat.

7-Bromo-(3a,5-cis)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethyl-pyrrolo[2,3- 6]indol-5-ol fumarate (HP 736) is a novel opioid analgesic. In vitro, HP 736 displaces [3H]dihydromorphine (IC50 = 8.3 x 10(-10) M) and [3H]bremazocine (IC50 = 7.4 x 10(-8) M) from mu and kappa opioid receptors, respectively, and displays modest acetylcholinesterase inhibitory activity (IC50 = 4.0 x 10(-5) M). The in vivo antinociceptive activity of HP 736 was found to be comparable to morphine in the modified Haffner's tail clip assay in mice and the D'Amour-Smith tail flick assay in rats. Moreover, these analgesic effects were found to be completely antagonized by the administration of the narcotic antagonist naloxone. A major liability of opioid analgesics such as morphine is the potential to cause cardiorespiratory depression. HP 736 (2, 4 and 10 mg/kg, i.v.) was found to cause significantly less respiratory depression in the anesthetized dog when compared to equivalent doses of morphine. At 10 mg/kg, morphine caused a 48% reduction in arterial oxygen partial pressure (PaO2) (-42.3 +/- 2.5 mm Hg) and a 52% increase in arterial carbon dioxide partial pressure (PaCO2) (21.0 +/- 3.4 mm Hg). In contrast, the same dose of HP 736 produced no significant decrease in PaO2, but did cause a slight 19% increase in PaCO2 (8.2 +/- 1.3 mm Hg), which was significantly less than the response seen after morphine treatment. It was found that pretreatment of the dogs with atropine sulfate (1 mg/kg, i.v.) "unmasked" the respiratory depressant activity of HP 736 (2 mg/kg, i.v.), indicating that the acetylcholinesterase inhibitory activity of the compound may contribute to its reduced cardiorespiratory liability. Finally, in confirmatory experiments conducted in conscious goats, HP 736 (0.5 mg/kg, i.v.) was found to stimulate pulmonary ventilation, increase PaO2 and oxygen consumption (+40%) and decrease PaCO2 with an overall stimulatory effect on the metabolic rate. In contrast, the same dose of morphine decreased metabolic rate, reduced pulmonary ventilation (-20%) and PaO2 and increased PaCO2. Overall, the results of these studies indicate that HP 736 is a potent opioid analgesic which appears to lack significant cardiorespiratory depressant activity.


Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.


Evidence for postjunctional release of ATP evoked by stimulation of muscarinic receptors in ileal longitudinal muscles of guinea pig.

The origin of the ATP release evoked by muscarinic agonists and veratridine from longitudinal muscles of the guinea pig was assessed with muscarinic antagonists. Acetylcholine (ACh) (1 microM) and bethanechol (10 microM) produced an immediate and marked ATP release, which was almost completely blocked by atropine (0.3 microM) and by the M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (1 microM); release was only slightly affected by tetrodotoxin (0.6 microM). The bethanechol-evoked release of ATP was partly, but not significantly, inhibited by pirenzepine, a M1 muscarinic antagonist. Veratridine, an ACh releaser, also elicited a delayed ATP release, in a concentration-related manner. This ATP release was greatly antagonized by atropine and by Ca++ removal from the medium, implying mediation by endogenous ACh released after depolarization. In contrast, electrically evoked ACh release was enhanced by atropine and 4-DAMP. Bethanechol, unlike veratridine, failed to elicit measurable ACh release from the tissue. The contraction evoked by bethanechol was notably inhibited by atropine and 4-DAMP, but not by pirenzepine and AFDX-116, a M2 muscarinic antagonist, at a concentration of 0.3 microM. These findings suggest strongly that ATP is postjunctionally released from the ileal smooth muscles after stimulation of postsynaptic muscarinic receptors, presumably M3 receptors.


Binding of typical and atypical antipsychotic agents to transiently expressed 5-HT1C receptors.

We determined the affinities of clozapine and 21 other typical and atypical antipsychotic agents for the cloned 5-hydroxytryptamine-1C (5-HT1C) receptor. For these studies, 5-HT1C receptors were transiently expressed in COS-7 cells using the vector pSVK3-5HT1C. We discovered that clozapine and several other putative typical and atypical antipsychotic agents (loxapine greater than tiosperone greater than SCH23390 greater than fluperlapine greater than rilapine greater than chlorpromazine) had relatively high affinities (7-30 nM) for the cloned 5-HT1C receptor. Other antipsychotic agents (risperidone greater than tenilapine greater than mesoridazine greater than thioridazine greater than cis-fluphenthixol) had intermediate affinities (30-100 nM), whereas many other antipsychotics (fluphenazine greater than spiperone greater than amperozide greater than melperone greater than thiothixene greater than haloperidol, metoclopramide, pimozide, domperidone, sulpiride) had low affinities (greater than 500 nM) for the cloned 5-HT1C receptor. The results indicate that although several putative atypical antipsychotic agents have high affinities for the cloned rat 5-HT1C receptor, the spectrum of drug binding does not correlate with the atypical nature of these compounds.


Somatostatin causes vasoconstriction, reduces blood flow and increases vascular permeability in the rat central nervous system.

Using radiolabeled microspheres, spinal cord blood flow was measured after spinal subarachnoid injections of 3.1- to 12.5-nmol doses of somatostatin through either indwelling i.t. catheters or acutely inserted intervertebral needles. With either injection technique, somatostatin caused significant dose-dependent reductions in thoracic and lumbosacral blood flow that could be partially blocked by a 5-min preinjection of the somatostatin receptor antagonist cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)], which has previously been shown to block the hindlimb flaccidity produced by these doses of somatostatin in conscious rats. The duration of these blood flow changes were appreciably less in the rats injected through indwelling i.t. catheters. Somatostatin-induced reductions in spinal cord perfusion were accompanied by transient pressor responses, reduced cardiac output, 3-fold increases in spinal cord cerebrospinal fluid lactic acid concentrations and breakdown of the blood-spinal cord barrier, as reflected by significantly increased extravasation of [125I]bovine serum albumin. By 24 hr postinjection, a 12.5-nmol dose of somatostatin caused appreciable spinal cord cellular injury, as evidenced by significant elevations in cerebrospinal fluid concentrations of lactate dehydrogenase. After topical application to exposed pial vessels of the parietal cortex, comparable doses of somatostatin caused immediate intense dose-related arteriolar vasospasm and subsequent extravasation of the visible macromolecular tracer Evans blue dye. We conclude that somatostatin has significant vasoconstrictory effects on the blood vessels of the brain and spinal cord of the rat that must be recognized and appreciated when studying its neuropharmacological actions in vivo.


Beta-blockers and depression. Evidence against an association.

To evaluate the relationship between beta-blockers and depression. Case-control study. New Jersey Medicaid recipients during July 1980 to December 1983. New depression case patients (N = 4302) were identified from Medicaid claims for depression markers (antidepressant drugs, in-hospital depression diagnosis, or electroconvulsive therapy). Control patients were randomly selected and matched on the basis of Medicaid enrollment on the case patients' date for first depression marker (index date), birth year, sex, race, and nursing home residency status. beta-Blocker use as evidenced by prescription claims in the year before the index date. Case patients overall were more likely to have taken beta-blockers (simple, matched odds ratio [OR] of 1.45; 95% confidence interval [CI], 1.29 to 1.62). Controlling for confounders (benzodiazepine use, frequent outpatient visits, and frequent use of medications other than beta-blockers) resulted in a null effect (OR = 0.98; 95% CI, 0.87 to 1.12). The ORs were consistently lower for case patients with a depression diagnosis or electroconvulsive therapy than for cases with only antidepressant use as a marker. These results did not vary by age, sex, race, nursing home status, or use of other selected specific medications. Ongoing beta-blocker use was not causally related to markers of depression. The difference between this study and those it contradicts is that this one identified certain confounding variables that accounted for the apparent relationship.


Spectrum of disease in persons with human immunodeficiency virus infection in the United States.

To describe the spectrum of disease in persons with human immunodeficiency virus (HIV) infection. Retrospective survey of medical records. More than 50 clinics, hospitals, and private medical practices in nine US cities. A total of 626 women and 7008 men 13 years of age or older with HIV infection who received medical care from January 1990 through March 1991 were consecutively enrolled. Any history of diseases in the 1987 case definition for the acquired immunodeficiency syndrome (AIDS), and during the 12-month period preceding enrollment (baseline period), the occurrence of other major diseases, hospitalizations, and results of CD4+ lymphocyte counts. Thirty-two percent of persons met the 1987 case definition for AIDS. The occurrence of an AIDS-indicator disease during the baseline period ranged from 3% (33/1011) to 46% (1254/2748) among persons with CD4+ lymphocyte counts of 0.50 x 10(9)/L or greater and fewer than 0.20 x 10(9)/L (greater than or equal to 500 and less than 200 CD4+ lymphocytes per microliter), respectively, and, at comparable CD4+ lymphocyte levels, was similar among women compared with men, and among persons who reported intravenous drug use compared with men who reported male-to-male sex. The frequency of one or more other major infectious diseases (eg, other pneumonias, bacterial sepsis, pulmonary tuberculosis) ranged from 6% to 16% among persons with CD4+ lymphocyte counts of 0.50 x 10(9)/L or greater and fewer than 0.20 x 10(9)/L, respectively; these illnesses were also associated with a history of intravenous drug use. Among persons who did not meet the 1987 AIDS case definition, 30% of those with an available CD4+ lymphocyte count had fewer CD4+ cells than 0.20 x 10(9)/L, 8% had one or more major infectious diseases, and 14% had one or more hospital admissions. For every person with AIDS at these sites, two additional persons with HIV infection were receiving medical care, many of whom had severe immunosuppression and a broad spectrum of serious HIV-related disease.


[Vecuronium-induced neuromuscular blockade in two patients with hyperparathyroidism and a patient with hypoparathyroidism].

Vecuronium-induced neuromuscular blockade was evaluated in two patients with primary hyperparathyroidism and in a patient with hypoparathyroidism. A 39 year old male with typical primary hyperparathyroidism was scheduled for surgical removal of the parathyroid adenoma. Serum levels of calcium and ionized calcium were 15.0 mg.dl-1 and 1.95 mmol.l-1, respectively. A 44 year old female suffering from primary hyperparathyroidism was also scheduled for surgical removal of the adenoma. Serum levels of calcium and ionized calcium were 12.5 mg.dl-1 and 1.51 mmol.l-1, respectively. A 63 year old male, suffering from postoperative secondary hypoparathyroidism and treated with calcium, was scheduled for surgical removal of the recurrent pharyngeal cancer. Serum levels of calcium and ionized calcium were 9.0 mg.dl-1 and 1.15 mmol.l-1, respectively. Anesthesia was induced with thiamylal 4-5 mg.kg-1 and vecuronium 0.08 mg.kg-1 and was maintained with 70% nitrous oxide in oxygen and fentanyl in all three patients. Neuromuscular blockade following the administration of vecuronium was measured by a big toe abduction evoked by supramaximal stimulation of the tibial nerve (Myograph 2000, Biometer, Denmark). In order to evaluate the effect of serum calcium level on vecuronium neuromuscular blockade, ten patients with normal serum levels of calcium, were examined in the same fashion. In only one patient with hyperparathyroidism, whose serum calcium was 15.0 mg.dl-1, the onset and the duration of vecuronium were later and shorter than those of other patients with normal serum levels of calcium. In conclusion, we should pay attention to the antagonistic responses to vecuronium in patients with severely high levels of serum calcium.


[A single bolus dose of vecuronium for rapid endotracheal intubation].

The changes in EMG evoked by train-of-four (TOF) stimulation of ulnar nerve were recorded to determine proper single bolus dose of vecuronium for endotracheal intubation in surgical patients. Onset and duration of neuromuscular block were judged by percent depression of EMG. Mean time intervals for 90% depression in TOF seen in 0.10 mg.kg-1 vecuronium group (n = 10), 0.15 mg.kg-1 vecuronium group (n = 10) and 0.20 mg.kg-1 vecuronium group (n = 10), were 181.1 sec, 135.0 sec and 120.0 sec, respectively. Similarly, mean time intervals for 100% depression for each vecuronium group were 240.0 sec, 177.5 sec and 160.0 sec, respectively. Onset time intervals in both 0.15 mg.kg-1 and 0.20 mg.kg-1 groups were significantly shorter than that in 0.10 mg.kg-1 group (P less than 0.05). On the other hand, mean time intervals for 25% recovery in EMG were 53.6 min in 0.10 mg.kg-1 group, 63.3 min in 0.15 mg.kg-1 group and 104.3 min in 0.20 mg.kg-1 group. No statistically significant difference was observed in recovery time between 0.10 mg.kg-1 and 0.15 mg.kg-1 group. These results indicate that the appropriate dose of vecuronium for rapid intubation is considered to be 0.15 mg.kg-1. This dose is allowable for surgical procedures of short duration.


[Adult T-cell leukemia with Ki-1 expression].

A 64-year-old man was admitted to our hospital complaining of fever and edema in February, 1990. Lymph node biopsy revealed diffuse lymphoma pleomorphic type according to the LSG classification. On hematological examination, leukocyte count was 23,500/microliters, of which 36% abnormal lymphocytes expressing CD2, CD3, CD4 and CD25 as same as the lymph node cells. Anti-HTLV-I antibody in serum was positive. From these data, the diagnosis of adult T-cell leukemia (ATL) was made. ATL cells in the blood and lymph node expressed CD30 (Ki-1). CD30 positive ATL cells derived from the blood was increased after short-term culture. The induction of Ki-1 antigen in cell lines and short-term cultured cells from ATL patients was accompanied by the appearance of the HTLV-I related antigen. Then, we suggest that there was some relation between expression of the Ki-1 antigen and activation by HTLV-I in ATL cells.


Antiviral agents.

In recent years, the antiviral armamentarium has expanded considerably. Currently available agents are virustatic, inhibiting specific steps in the process of viral replication. No agent is active against nonreplicating or latent viruses. Acyclovir is useful in the treatment of genital herpes, herpes simplex encephalitis, mucocutaneous herpetic infection, varicella infection in the immunosuppressed host, and herpes zoster infection in the normal and the immunosuppressed host. It can also be used for prevention of herpesvirus infection in immunocompromised patients. Ganciclovir is indicated for the treatment of cytomegalovirus retinitis in patients with acquired immunodeficiency syndrome (AIDS) and is effective in the management of organ-specific cytomegalovirus infection in other immunocompromised patients. Chronic hepatitis C and condyloma acuminatum due to human papillomavirus respond to therapy with interferon alfa-2b. Patients with human immunodeficiency virus infection and CD4 lymphocyte counts of less than 500 cells/mm3 should be treated with zidovudine. Amantadine is useful in a therapeutic and prophylactic role in the management of influenza A virus infection. With the expanded use of and indications for antiviral therapy, clinically significant resistance to these agents has been encountered with increasing frequency.


Antituberculous agents.

Antituberculous agents have radically improved the prognosis of patients with active tuberculosis. Generally, 6-month and 9-month antituberculous regimens have been successful, and surgical therapy is rarely needed. Extrapulmonary tuberculosis should be managed with the same drug regimens as pulmonary tuberculosis. The major cause of therapeutic failure is poor compliance of the patient in taking the prescribed medication regularly. A second cause of failure of treatment is resistance of tubercule bacilli to antimicrobial agents used. When failure of treatment is apparent, careful reassessment by physicians experienced in the treatment of tuberculosis is indicated. A single drug should never be added to a failing regimen. Isoniazid administered prophylactically for 6 to 12 months is effective in most cases.


Fibrinogen genotype and risk of peripheral atherosclerosis.

There is conflicting evidence about the influence of fibrinogen genotype on plasma fibrinogen concentrations, and the relation between genotype and atherosclerotic disease has not been studied. In a population-based case-control study we aimed to find out whether certain fibrinogen genotypes are associated with an increased risk of peripheral atherosclerosis. 121 subjects with peripheral arterial disease and 126 healthy controls matched for age and sex were selected from a random population sample aged 55-74 years in the Edinburgh Artery Study. Mean fibrinogen concentrations were higher in cases than in controls (3.12 [95% confidence interval 2.99-3.26] vs 2.75 [2.64-2.85], p less than 0.001). A greater proportion of cases than controls were homozygous or heterozygous for an allele at the beta fibrinogen locus (4.2 kb allele, Bcl I digestion); the allele frequency was 0.197 in cases and 0.097 in controls (p less than 0.005). Extended haplotypes for 4.2 kb heterozygotes were also associated with an increased risk of peripheral arterial disease. However, haplotype had only a small effect on the association of plasma fibrinogen concentration with disease, and the relation of haplotype with disease was independent of age, sex, social class, smoking status, plasma fibrinogen, alcohol consumption, body mass index, and diabetes mellitus. We conclude that variation at the beta fibrinogen locus is associated with an increased risk of peripheral atherosclerosis. The influence is not mediated simply by way of increased fibrinogen concentrations but could be due to a structurally variant fibrinogen or linkage disequilibrium with a neighbouring gene.


Voluntary dehydration and heat intolerance in cystic fibrosis.

Although exercise may be beneficial in cystic fibrosis (CF), patients' low tolerance to climatic heat stress means that physical exertion can increase morbidity and mortality. We postulated that the high salt content of CF patients' sweat and the consequent absence of body-fluid hyperosmolality during a long episode of sweating might deprive such patients of a thirst stimulus. Eight children with CF (four boys, four girls; aged 9.5-14.1 years) and eight controls, matched for age and sex, attended two randomly ordered sessions of exercise (cycling) in a chamber at 31-33 degrees C, relative humidity 43-47%. 20 min bouts of exercise (at 45% of predetermined maximum oxygen uptake) were interspersed with 25 min rest periods. At one session, chilled water was given every 15-20 min to replace fluid lost; at the other, drinking was guided by the child's thirst. At the thirst-guided session, CF patients drank much less than the controls did (0.80% vs 1.73% initial body weight) and lost twice as much fluid (1.57% vs 0.78% initial body weight). The recovery of heart rate after exercise was slower in CF patients, but there were no other signs of heat strain. The groups did not differ in any variable during the forced drinking session. We conclude that children with CF underestimate their fluid needs and undergo excessive dehydration during extended exposure to hot conditions.


Pulmonary toxic effects of continuous desferrioxamine administration in acute iron poisoning.

The drug of choice for the treatment of iron poisoning is desferrioxamine, though the best route of administration, dose, and duration of treatment are unclear. We report fatal lung injury in four patients who were treated with continuous intravenous infusions. The patients, aged 19-26 years, had received desferrioxamine infusions of 15 mg/kg per h for 65-92 h. Respiratory distress developed after 32-72 h. The patients met clinical, physiological, and necropsy criteria for the diagnosis of adult respiratory distress syndrome (ARDS); none had any of the known risk factors for the development of this disorder. We reviewed the records of forty-three iron-poisoned patients treated with desferrioxamine infusions. No patient treated for less than 24 h had pulmonary complications; however, of the fourteen treated for longer than 24 h, four were the patients with ARDS and four others had pulmonary oedema of other causes. We suggest that the pulmonary complications are caused by continuous infusion of desferrioxamine and that the ARDS in these patient was a consequence of free-radical generation. We recommend that desferrioxamine infusion should not be administered for longer than 24 h.


Relation between coronary risk and coronary mortality in women of the Renfrew and Paisley survey: comparison with men.

Most epidemiological and intervention studies in patients with coronary artery disease have focused on men, the assumption being that such data can be extrapolated to women. However, there is little evidence to support this belief. We have completed a fifteen-year follow-up of 15,399 adults, including 8262 women, who lived in Renfrew and Paisley and were aged 45-64 years when screened between 1972 and 1976. We identified 490 deaths from coronary heart disease (CHD) in women and 878 in men. Women were more likely to have high cholesterol, to be obese, and to come from lower social classes than men, but they smoked less and had similar blood pressures. The relative risk--top to bottom quintile (95% Cl)--of cholesterol for coronary death after adjustment for all other risk markers was slightly greater in women (1.77 [1.45,2.16]) than in men (1.56 [1.32, 1.85]), but absolute and attributable risk were lower. Thus, women in the top quintile for cholesterol had lower coronary mortality (6.1 deaths per thousand patient years) than men in the bottom quintile (6.8 deaths per thousand patient years). Moreover, it was estimated that there would have been only 103 (21%) fewer CHD deaths in women, yet 211 (24%) fewer in men, if mortality had been the same for women and men in the lowest quintiles of cholesterol. Trends showing similar relative risks in these women, but lower absolute and attributable risks than in men, were present for smoking, diastolic blood pressure, and social class. There was no relation between obesity and coronary death after adjustment for other risks. Our results suggest that some other factors protect women against CHD. The potential for women to reduce their risk of CHD by changes in lifestyle may be less than for men.


Glomerulonephritis with end-stage liver disease in childhood.

Renal biopsies were done perioperatively in 18 children receiving liver grafts. All specimens showed glomerulonephritis, which was mesangial-proliferative in 15 and mesangio-capillary in 3. Of the 16 children who were alive four or more months after transplantation, only 1 showed progressive deterioration of renal function; 1 other had a subnormal but static glomerular filtration rate. In all 6 children who had proteinuria before operation, the urine became normal.


Gene therapy for cancer.

The molecular basis of cancer is now understood to involve activation of dominant oncogenes and inactivation of tumour suppressor genes, and these genetic events may represent novel targets for cancer therapy. This review focuses on the potential use and ethical implications of gene transfer to alter the behaviour of somatic cells in cancer patients. Antisense nucleic acids and ribozymes represent informational drugs that may be used to modulate the expression of selected genes and suppress malignant behaviour in cancer cells. Genetic immunomodulation by introducing genes for cytokines into cancer cells or lymphocytes can stimulate a cytotoxic immune response against the tumour. Gene transfer techniques can be applied to target prodrug activation specifically to tumour cells and also to protect normal tissues against toxic chemotherapy. Gene replacement therapy could even be used to restore the function of defective tumour suppressor genes.


Low serum cholesterol and suicide.

Primary prevention trials which have shown that the lowering of serum cholesterol concentrations in middle-aged subjects by diet, drugs, or both leads to a decrease in coronary heart disease have also reported an increase in deaths due to suicide or violence. There has been no adequate explanation for this association. I have reviewed the relevant published work and describe a physiological mechanism that might account for this curious finding. One of the functions of serotonin in the central nervous system is the suppression of harmful behavioural impulses. When mouse brain synaptosomal membrane cholesterol is increased there is a pronounced increase in the number of serotonin receptors. Low membrane cholesterol decreases the number of serotonin receptors. Since membrane cholesterol exchanges freely with cholesterol in the surrounding medium, a lowered serum cholesterol concentration may contribute to a decrease in brain serotonin, with poorer suppression of aggressive behaviour.


Cyclic AMP accumulation alters calmodulin localization in SK-N-SH human neuroblastoma cells.

In SK-N-SH human neuroblastoma cells, the muscarinic agonist carbachol promotes polyphosphoinositide (PPI) hydrolysis via M3 receptors and increases cyclic AMP levels through an unidentified mechanism. Activation of PPI hydrolysis by carbachol elicits a robust translocation of CaM from membranes into cytosol which was previously shown to be mimicked by the addition of the calcium ionophore ionomycin and the phorbol ester TPA28. The effect of agonist-stimulated second messenger production on CaM localization was determined by activating receptors that increase and decrease adenylyl cyclase activity on SK-N-SH cells. VIP (10 microM), prostaglandin E1 (30 microM) and forskolin (10 microM) all increased adenylyl cyclase activity 8- to 10-fold above the activity with 1 microM GTP. Carbachol (100 microM) did not stimulate adenylyl cyclase activity. The alpha 2-adrenergic agonist UK 14,304 (0.1 microM) and the delta and mu opioid DPDPE (10 microM) and DAMGO (10 microM) inhibited forskolin-stimulated cyclic AMP formation by 27-32%. CaM did not stimulate adenylyl cyclase activity. Incubation of cells with vasoactive intestinal polypeptide (VIP), dibutyryl cyclic AMP and forskolin, resulted in 30% decrease in membrane CaM and an increase in cytosolic CaM of 40-50%. The CaM translocation with the combination of an agent that elevates cyclic AMP levels and a low dose of carbachol was not different from that observed with either agent alone. UK 14,304, DPDPE and DAMGO potentiated carbachol-stimulated increases in cytosolic CaM. Upon the addition of carbachol, a 5-fold increase in intracellular calcium concentration measured with fura-2 fluorescence was observed. VIP and UK 14,304 elevated intracellular calcium concentrations 2 to 3 fold, while forskolin (10 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential induction of immediate early genes by excitatory amino acid receptor types in primary cultures of cortical and striatal neurons.

In primary cultures of neurons from cerebral cortex and striatum, 30 s stimulation with the excitatory amino acid glutamate elicited a 5 to 9-fold increase in immediate early gene (IEG) mRNAs. Glutamate increased c-fos, c-jun, jun-B, and NGFI-A (zif/268) mRNAs by binding to both alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor types, and increased c-fos, jun-B, and NGFI-A mRNAs by binding to the metabotropic receptor. NMDA receptor activation elicited IEG expression by a transmembrane calcium influx; AMPA receptor-induced depolarization played a permissive role for the opening of the NMDA receptor channel. The protein kinase C (PKC) inhibitor H-7 (but not inhibitors of cyclic nucleotide-dependent and calcium/calmodulin-dependent protein kinases) partially blocked IEG expression induced by glutamate.


The effects of excitatory amino acids on proenkephalin and prodynorphin mRNA levels in the hippocampal dentate gyrus of the rat; an in situ hybridization study.

Injection of N-methyl-D-aspartate (NMDA, 7.5 micrograms) kainate (1 microgram) or quisqualate (2 micrograms) into the rat dorsal hippocampus induced wet-dog shakes and convulsions. As shown by an in situ immunohistochemical analysis, 3 h after the excitatory amino acids injections the rats displayed a bilateral profound elevation of the proenkephalin and prodynorphin mRNA levels in dentate gyrus granule cells (2-3 or 1.5-2 fold higher than control levels, respectively). Pretreatment of rats with D-amino-phosphonovalerate (D-APV, 10 micrograms), a selective antagonist of NMDA receptor, prevented the behavioral and biochemical changes evoked by NMDA. The changes in the behavior and gene expression evoked by kainate or quisqualate were diminished in rats which received 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX, 2 micrograms), a putative antagonist of quisqualate and kainate receptors. The study demonstrated that activation of NMDA, quisqualate or kainate receptors in the hippocampus induced seizures associated with a marked increase in the proenkephalin (PENK) and the prodynorphin (PDYN) gene expression in the rat dentate gyrus.


Cellular localization of tyrosine hydroxylase mRNA and cholecystokinin mRNA-containing cells in the ventral mesencephalon of the common marmoset: effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

In situ hybridization histochemistry was used to localize tyrosine hydroxylase (TH) mRNA and cholecystokinin (CCK) mRNA-expressing cells in the ventral mesencephalon of the common marmoset (Callithrix jacchus) and to examine the effects of the dopaminergic (DA) neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on these two populations of neurons in the pars compacta of the substantia nigra (SNc) and ventral tegmental area (VTA). X-ray film and liquid emulsion autoradiography of brain sections hybridized with an 35S-labelled synthetic 45-mer antisense human TH oligonucleotide probe showed strong hybridization signals and dense populations of TH mRNA expressing cells in the SNc and VTA at all levels, in the control marmoset brain. In the MPTP-treated brain, there was a substantial reduction of TH mRNA in the ventral midbrain. The loss of TH mRNA-expressing cells amounted to 98% in the lateral SNc, 88% in the medial SNc and 33% in the VTA. In situ hybridization of adjacent sections with an 35S-labelled synthetic 45-mer antisense human CCK oligonucleotide probe showed a weak hybridization signal for CCK mRNA in the ventral midbrain of the control brain. Emulsion autoradiography demonstrated CCK mRNA expressing cells in the SNc and VTA at all levels with the number of cells in the VTA similar to that for TH mRNA. However, the number of cells in the SNc expressing CCK mRNA was a fraction (1/4) of that expressing TH mRNA; moreover, the level of expression per cell was substantially less than that for TH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)


Immune-mediated cytopenia following bone marrow transplantation. Case reports and review of the literature.

We describe 3 cases of immune-mediated cytopenia occurring after bone marrow transplantation (BMT). In 1 case, only the platelet line was affected, whereas in the other 2 cases more than 1 cell lineage was involved simultaneously. Two of the cases presented with falling peripheral blood counts following apparently normal early engraftment, while in 1 of the cases the affected lineage failed to appear in the peripheral blood despite normal engraftment of the other lineages. In all 3 cases the cytopenia improved following the initiation of treatment with systemic corticosteroids. Immune-mediated cytopenia following bone marrow transplantation may occur via alloimmune or autoimmune mechanisms. Alloimmune cytopenias have arisen in the context of major or minor mismatches in the ABO system, but cases related to mismatches in the Rh system and other erythroid and non-erythroid alloantigen systems may also occur. Alloimmune cytopenias have been reported primarily in the setting of allogeneic BMT, whereas autoimmune cytopenias have been reported following both allogeneic and autologous BMT. Immune-mediated cytopenia may present as early as the day of transplant, or as late as many months afterward. The possibility of immune-mediated cytopenia should always be considered when unexpected peripheral blood cytopenia is present, or when unexpected hemolysis develops, following bone marrow transplantation. The diagnosis is supported by a normal appearance of the affected lineage or lineages in the bone marrow, the absence of other apparent causes for the cytopenia, and the presence of the relevant auto- or allo-antibodies in the serum. However, any of these features may be absent in individual cases. The importance of these syndromes lies in the fact that they may be life-threatening, yet they often respond well to steroids or other standard immunosuppressive measures. It is important to be aware that effective prophylactic measures are available for patients receiving ABO- or Rh-incompatible marrow.


Isolation, characterization, and genetic analysis of monosomic, aneuploid mutants of Candida albicans.

A white, prototrophic Candida albicans strain, heterozygous for the ADE2 gene (ade2/ADE2), was treated with the antimitotic agent methyl benzimidazole carbamate, and yielded red, adenine-requiring colonies at a rate of 4 x 10(-3), an order of magnitude higher than the spontaneous rate of Ade- colony formation. These red Ade- colonies were small, growing at approximately half the rate of the parent strain, and gave rise to large red colonies spontaneously. When the chromosomes of the small red colonies were separated by pulsed-field gel electrophoresis, the band hybridizing with the ADE2 gene was diminished in staining intensity by half relative to the parent and large red-colony strains. Restriction fragment-length polymorphism analysis and auxotrophic mutant spectra after mutagenesis suggested that the small red Ade- strains were monosomic aneuploids lacking one of a pair of chromosome homologues, while the large red strains had regained a homologue, presumably via a second non-disjunction event. Parasexual genetic analysis of two of the auxotrophs isolated from a putative aneuploid suggested that both mutations were linked to the ADE2 gene. These experiments suggest that targeted chromosome loss and monosomic, aneuploid strains have the potential to extend the scope of genetic analysis in this diploid, asexual organism.


Silent pilin genes of Neisseria gonorrhoeae MS11 and the occurrence of related hypervariant sequences among other gonococcal isolates.

Pilin variation in Neisseria gonorrhoeae depends on a family of variant genes that undergo homologous, intragenic recombination. This work focuses on the repertoire of silent variant pilin genes in strain MS11, which contribute to the extensive variation of the expressed gene copy. A total of 17 silent copies were identified, which are, to varying degrees, truncated at their 5' coding region and grouped in seven distinct pil loci. Most silent copies belong to loci pilS1, pilS2 and pilS6, which contain six, two and three silent copies, respectively, tandemly arranged. The pilS5 and pilS7 loci each contain only a single copy. In addition, two silent copies are associated with each of the two pilE loci. By comparison with sequences present in the expressed gene of other variants of the same strain, it is suggested that each silent locus is capable of donating variant sequences into the expression locus and, thus, each silent copy can contribute to the variability of pilin expression. Often, concomitant with changes in the expressed copy, the silent copies of the pilE1 locus undergo recombinations as well. Analyses of unrelated clinical isolates of N. gonorrhoeae reveal homologies of hypervariant pilin sequences with those present in strain MS11, suggesting a limited diversity of such sequences within the gonococcal population and the existence of substantial functional constraints on the variability of pilin and pili. The data further indicate that hypervariant pilin sequences are subject to horizontal exchange and interstrain recombination.


Linking prescription and patient-identifying data: a pilot study.

To link a Pharmaceutical Benefits Scheme (PBS) prescription data set with patient-identifying data held by the Health Insurance Commission (HIC) and to then determine the prevalence of prescribing of cardiovascular drugs, non-steroidal anti-inflammatory drugs, hypnotics and minor tranquillizers, and diuretics (prescribed without other cardiovascular drugs) by age and sex in two defined populations. Prescription data for a three-month period in 1985 were matched with patient-identifying information to obtain a database which included the patient's age, sex and an identifying number, for each prescription record. The percentages of the population taking a drug from each of the drug categories mentioned above were then determined and the effect of age, sex and region of residence on prescribing prevalence was investigated using logistic regression analysis. Two rural regions of Australia with a total population of 65,087 residents. Of the 101,383 prescriptions dispensed over the period 96% could be matched with HIC information. In the two regions combined, the percentages of the population taking a cardiovascular drug, non-steroidal anti-inflammatory drug, hypnotic or minor tranquilizer, or diuretic (without other cardiovascular drugs) were 10.4%, 5.8%, 3.5% and 2.5%, respectively. Prescribing rates were higher for females than males, increased with age and varied between the two regions. Approximately 5% of women aged 30-39 years were taking diuretics without other cardiovascular drugs, compared with only 0.2% of men in the same age group. This pilot study illustrates the use of a patient-identified prescription database for drug utilisation review, therapeutic audit and hypothesis generation.


Gi alpha 1 selectively couples somatostatin receptors to adenylyl cyclase in pituitary-derived AtT-20 cells.

Somatostatin (SRIF) receptors are coupled to the catalytic subunit of adenylyl cyclase via pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). To identify which G proteins link SRIF receptors to adenylyl cyclase, G(o) alpha, Gi alpha, and its different subtypes were individually blocked in AtT-20 cell membranes with G alpha subtype-selective antisera. Antiserum directed against the carboxyl-terminal region of Gi alpha blocked SRIF inhibition of forskolin-stimulated adenylyl cyclase activity, and this effect was prevented by the peptide to which the antiserum was generated. However, antiserum directed against the carboxyl-terminal region of G(o) alpha did not affect SRIF inhibition of adenylyl cyclase activity, indicating that Gi alpha couples SRIF receptors to adenylyl cyclase but G(o) alpha does not. Peptide-directed antisera against Gi alpha 1 completely blocked SRIF inhibition of adenylyl cyclase activity. In contrast, antisera directed against either Gi alpha 2 or Gi alpha 3 did not affect the actions of SRIF. The results of these studies indicate that Gi alpha 1 selectively couples SRIF receptors to the catalytic subunit of adenylyl cyclase in AtT-20 cell membranes. Because previous studies have shown that SRIF receptors are able to couple to Gi alpha 1, Gi alpha 3, and G(o) alpha, the results suggest that different G proteins may specify the coupling of SRIF receptors to distinct cellular effector systems.


Vasoactive intestinal polypeptide facilitates tyrosine hydroxylase induction by cholinergic agonists in bovine adrenal chromaffin cells.

The possibility that vasoactive intestinal polypeptide (VIP) may facilitate the nicotine-mediated induction of adrenal medullary tyrosine hydroxylase (TH) was investigated with primary cultures (5-7 days in vitro) of bovine adrenal chromaffin (BAC) cells. Exposure of BAC cells to 100 microM nicotine led to only a marginal increase in the amount of TH mRNA, TH protein, and TH activity. VIP, alone or in the presence of a phosphodiesterase inhibitor, produced a marked increase in TH mRNA, TH protein, and TH activity. Moreover, VIP together with nicotine, at concentrations that alone were devoid of effect, increased the amount of TH mRNA and TH activity. A synergistic effect of VIP and nicotine on cAMP accumulation in BAC cells was also apparent. The marginal effects of large doses of nicotine on both cAMP accumulation and TH induction were blocked completely by hexamethonium but were also partially inhibited by the VIP antagonist [p-chloro-D-Phe6,Leu17]-VIP. Nicotine may, therefore, stimulate the release of VIP from cultured BAC cells and VIP, in turn, by increasing cAMP, may synergize with nicotine to enhance TH gene expression.


Distinct regulation of beta 1- and beta 2-adrenergic receptors in Chinese hamster fibroblasts.

The agonist-induced reduction of beta-adrenergic receptor (beta AR) cell surface density is a well documented phenomenon. The mechanisms responsible for this regulation have been well characterized for the beta 2AR. They include a rapid sequestration of the receptor away from the cell surface in a vesicular compartment and a longer term down-regulation of the total beta 2AR number. In contrast, very little is known about the cell surface regulation of the beta 1AR. In the present study, we have compared the agonist-mediated regulation of beta 1- and beta 2AR in Chinese hamster fibroblasts transfected with the cDNA encoding either beta AR subtype. Cells expressing similar numbers of the two beta AR subtypes were selected for the study. The expressed receptors exhibit typical beta 1- and beta 2AR selectivity for agonists and antagonists, as assessed by radioligand binding. Both receptors were found to be positively coupled to the adenylyl cyclase stimulatory pathway, but marked differences in the receptor regulation profiles were observed. Treatment of the cells expressing the beta 2AR with the agonist isoproterenol leads to a rapid sequestration of greater than 30% of the receptors away from the cell surface into a light vesicular fraction, where they are inaccessible to the hydrophilic ligand CGP-12177. In contrast, virtually no agonist-induced sequestration is observed in the cells expressing the beta 1AR. Longer exposure of the cells to isoproterenol leads to a time-dependent reduction in the total number of beta ARs in both beta 1- and beta 2AR-expressing cell lines. However, this down-regulation is significantly slower in the cells expressing the beta 1AR. In fact, no appreciable down-regulation of the beta 1ARs is detected in the first 4 hr of agonist treatment, compared with a down-regulation of greater than 50% of the beta 2ARs for the same period. After a 24-hr treatment with isoproterenol, less than 20% of the original number of beta 2ARs remain, whereas 60% of the beta 1ARs are still present after the same treatment. These results, therefore, suggest that, when expressed in an identical cell line, beta 1AR and beta 2AR follow distinct patterns of regulation. In fact, both agonist-induced sequestration and down-regulation are considerably blunted for the beta 1AR, compared with the beta 2AR.


Cyclic AMP response element-binding protein and the catalytic subunit of protein kinase A are present in F9 embryonal carcinoma cells but are unable to activate the somatostatin promoter.

The cyclic AMP (cAMP) response elements (CREs) of the somatostatin and vasoactive intestinal peptide (VIP) promoters contain binding sites for CRE-binding protein (CREB) that are essential for cAMP-regulated transcription. Using F9 embryonal carcinoma cells, we show that the somatostatin and VIP promoters exhibit a differentiation-dependent cAMP response, demonstrating that these promoters are regulated by transcription factors that become active during differentiation. Lack of cAMP responsiveness of the somatostatin promoter in undifferentiated cells is not due to the absence of known positive-acting factors (the catalytic subunit of protein kinase A [cPKA] and CREB) or a general inhibition of protein kinase A activity. Since overexpression of exogenous cPKA and CREB is sufficient to activate the somatostatin promoter in undifferentiated cells, these findings suggest that a negative factor(s) represses endogenous cPKA and CREB. In contrast to their effects on somatostatin, exogenous CREB and cPKA do not activate the VIP promoter. Thus, despite coregulation during differentiation and the ability to bind CREB, the somatostatin and VIP promoters are not coordinately activated by CREB in undifferentiated F9 cells.


Restriction site generating-polymerase chain reaction (RG-PCR) for the probeless detection of hidden genetic variation: application to the study of some common cystic fibrosis mutations.

In this report we describe the use of a DNA amplification technique in which modified primers introduce a base substitution adjacent to the codon of interest and create an artificial restriction site for the detection of mutations which do not produce or modify a naturally occurring restriction site (restriction site generating-polymerase chain reaction, RG-PCR). RG-PCR was developed and applied to the screening in an Italian population sample of several relatively common cystic fibrosis mutations which are not amenable to analysis with a known restriction endonuclease: G542X, 2869insG, Y913C, N1303K, and 1717-1GA. This method, which allows the identification of virtually any single base change by restriction enzyme analysis and without the need for molecular probes, is rapid and easy to perform. The combined use of RG-PCR for several different CF mutations in multiplex tests further expands the advantages of this approach.


Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer.

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.


In vivo modulation of N-methyl-D-aspartate receptor-dependent long-term potentiation by the glycine modulatory site.

The role of the glycine modulatory site in N-methyl-D-aspartate receptor function was examined by determining the effect of the glycine site antagonist, 7-chlorokynurenic acid, on the induction of long-term potentiation at the commissural-CA1 synapse in anesthetized rats. Robust long-term potentiation of population excitatory postsynaptic potentials and population spike responses recorded extracellularly in the stratum pyramidale and in stratum radiatum of CA1 developed after high frequency stimulation (100 Hz for 1 s) of commissural fibers during continuous intrahippocampal administration of vehicle solution (0.15 M NaCl). In contrast, infusion of either 7-chlorokynurenic acid (400 microM) or of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonovaleric acid (100 microM), significantly attenuated or completely blocked the development of long-term potentiation. When 7-chlorokynurenic acid was infused together with the glycine analog, D-serine (1 mM), long-term potentiation developed that was comparable to that observed in control animals. Intrahippocampal administration of D-serine alone was associated with slightly greater magnitude of long-term potentiation than observed in control animals. Collectively, these findings establish that in intact hippocampus, activity at the glycine modulatory site is necessary for activation of the N-methyl-D-aspartate receptor complex. Furthermore, these results suggest that the glycine modulatory site may not be fully saturated in vivo, and thus can serve to regulate N-methyl-D-aspartate receptor function.


Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum.

An electron microscopic, double-labelling immunocytochemical procedure was used to assess the level of fixed glutamate and glutamine in different cell profiles in ultrathin sections of rat cerebellar cortex. The procedure was based on sequential immunolabelling with two rabbit antisera, using gold particles of different sizes as markers and formaldehyde vapour as a means to avoid interference between the two incubations. Model sections containing a series of known concentrations of the respective amino acids (aldehyde--fixed to rat brain protein) were incubated together with the tissue material. These revealed a close to linear relationship between gold particle density and antigen concentration throughout the range of biological relevance. The ratio between the density of the two categories of gold particles was calculated for the individual profile types. This ratio showed a 20-fold variation, with the highest glutamate/glutamine ratios obtained for putative excitatory terminals (terminals of parallel fibres in the outer part of the molecular layer, followed by mossy and climbing fibre boutons) and the lowest for glial cells (Bergmann glia, astrocytes in the granule cell layer, and oligodendrocytes). Granule cell bodies and dendrites, and cell bodies and processes of putative GABAergic cells (Purkinje, basket and Golgi cells) displayed intermediate ratios. The ratios corresponded to millimolar ratios (mM fixed glutamate/mM fixed glutamine) ranging from 4.5 to 0.2, tentatively assessed by adjusting for differences in labelling efficiency of the two antigens. Our results show that the compartmentation of glutamate and glutamine, an issue previously addressed mainly in the test tube, can be studied in morphologically intact preparations at a resolution that matches the complexity of CNS tissue. The data indicate that glutamate is effectively converted to glutamine in all categories of glial cells, and that glutamate synthesis prevails in each of the three types of excitatory terminals in the cerebellar cortex. Terminals of putative GABAergic cells form a distinct low glutamate/low glutamine compartment.


Brainstem excitatory amino acid receptors in nociception: microinjection mapping and pharmacological characterization of glutamate-sensitive sites in the brainstem associated with algogenic behavior.

In awake, freely moving rats, the intracerebral administration of the excitatory amino acid L-glutamate (30 nmol/0.5 microliters) into discrete regions of the brainstem resulted in a transient and spontaneous pain-like syndrome characterized by an initial vocalization and vigorous escape behavior. Systematic microinjection mapping studies were carried out at sites distributed caudally from the lower medulla and rostrally into diencephalon. These studies revealed that the spontaneous pain-like behavior was observed to occur after glutamate injection in 13% of 331 microinjected sites, and these sensitive sites were largely limited to the mesencephalic periaqueductal gray matter. The behavioral syndrome was dose-dependent and antagonized in a dose-dependent fashion by the glutamate receptor antagonists MK 801 and DL-2-amino-5 phosphonovalerate but not by gamma-D-glutamyl-amino-methylsulfonic acid. The pain-like behavior was also produced by the other excitatory amino acid receptor agonists N-methyl-D-aspartate, quisqualate and to a certain extent by kainate in a dose-dependent manner with the order of potency being N-methyl-D-aspartate = kainate greater than quisqualate greater than D-glutamate. The effects of N-methyl-D-aspartate and quisqualate were antagonized by MK 801 and DL-2-amino-5 phosphonovalerate but not by gamma-D-glutamyl-amino-methylsulfonic acid. It is suggested that the pain-like behavioral syndrome is the result of focal occupation of N-methyl-D-aspartate receptors on neuronal populations in the terminal regions of rostrally projecting spinomesencephalic systems.


Tachykinins and calcitonin gene-related peptide as co-transmitters in local motor responses produced by sensory nerve activation in the guinea-pig isolated renal pelvis.

Electrical field stimulation of circular muscle strips from the guinea-pig isolated renal pelvis produces a frequency-dependent positive inotropic effect of the spontaneous contractions which is unaffected by atropine and guanethidine and abolished by tetrodotoxin or in vitro capsaicin desensitization. Omega conotoxin fraction GVIA markedly inhibited the response to low frequencies of stimulation but had only a partial or minor inhibitory effect at higher frequencies. Tachykinins produce a concentration-dependent positive inotropic effect, neurokinin A being more potent than substance P. On the other hand, rat alpha calcitonin gene-related peptide (CGRP) inhibited spontaneous contractions of the renal pelvis. MEN 10,376 a neurokinin A (4-10) analog, antagonized the positive inotropism produced by neurokinin A, without affecting the response to KCl, and suppressed the positive inotropic response produced by electrical field stimulation. In the presence of MEN 10,376, a negative inotropic response was produced by electrical field stimulation which was antagonized by the C-terminal fragment (8-37) of human alpha calcitonin gene-related peptide (hCGRP). hCGRP (8-37) antagonized the negative inotropic effect of exogenously administered CGRP without affecting inhibition by isoprenaline. Application of capsaicin (10 microM) produced a marked increase in the outflow of substance P-, neurokinin A- and CGRP-like immunoreactivities from the superfused guinea-pig renal pelvis. Substance P-, neurokinin A- and CGRP-like immunoreactivities were also detected in tissue extracts of the renal pelvis by radioimmunoassay. These experiments indicate that peptide release from peripheral endings of capsaicin-sensitive primary afferents represents the major type of nerve-mediated response affecting motility of the guinea-pig isolated renal pelvis. Tachykinins and CGRP act as physiological antagonists and the excitatory action of tachykinins prevails over the inhibitory action of CGRP. Local modulation of renal pelvis motility by sensory nerves could facilitate removal of irritants present in the urine, protecting the kidney during obstruction and ureteral antiperistalsis.


Analysis of extrathalamic synaptic influences on reactions of sensorimotor cortical neurons during conditioning.

The effects of iontophoretic application of acetylcholine, noradrenaline, serotonin and their blockers on neuronal activity were studied in the cat before and during fulfillment of conditioned instrumental placing reflex. It was found that acetylcholine increased the background neuronal activity through muscarinic cholinergic receptors and noradrenaline decreased it through beta-adrenoceptors in a considerable proportion of the cortical neurons. Serotonin had no reliable effect on the background activity. At the same time, it facilitated an initial component of the impulse reaction to conditioned stimulus and part of the impulse reaction preceding the start of the conditioned movement. Acetylcholine applied iontophoretically also facilitated the evoked responses in some cortical neurons via nicotinic cholinergic receptors. On the contrary, iontophoretic application of noradrenaline or ephedrine decreased the evoked activity of some neurons. Application of beta-adrenergic receptor blocker, propranolol, led to an increase of neuronal responses to conditioned stimuli. Evidently, noradrenergic projections exert a steady inhibitory influence on the cortical neurons during natural functioning of the cortex. It is concluded that cortical reactions evoked by activation of thalamic projections and intracortical connections are modulated and regulated by extrathalamic projections to the cortex.


Long-term management of the patient with stable angina.

Advances in the pharmacologic, medical, and surgical treatment of ischemic heart disease have led to the recognition of new goals in the treatment of patients with stable angina, including the relief of symptoms, treatment of underlying causes, and the enhancement of their quality of life. As research continues to provide more information about the pathophysiologic process of ischemic heart disease, new procedures, diagnostics, and management techniques will continue to emerge. In considering the long-term management of patients with stable angina, the primary role of the nurse is in providing relevant information regarding the management of anginal symptoms and related lifestyle modifications. Integrating knowledge of the disease process and its treatment into nursing practice will achieve a comprehensive plan of nursing care that addresses the physiologic, psychosocial, and educational needs of the patient and family members.


Drug treatment of COPD. Controversies about agents and how to deliver them.

Anticholinergics (in particular, ipratropium bromide [Atrovent]) are first-line therapy in patients with chronic obstructive pulmonary disease (COPD). Although more studies are needed to support the use of combination therapy, adding an inhaled beta agonist to the therapeutic regimen is reasonable in patients who remain symptomatic and need quick relief. Patients frequently receive inadequate amounts of drug with standard doses delivered by metered-dose inhalers, often as the result of improper technique, so symptomatic patients may require higher doses. Caution is recommended when the dose of inhaled sympathomimetics is increased in COPD patients with ischemic heart disease or tachyarrhythmias. The addition of an oral sympathomimetic is seldom necessary. Theophylline may be considered in outpatients who remain symptomatic despite their use of inhaled bronchodilators, but heart disease, seizure disorders, and gastroesophageal reflux are contraindications. Corticosteroid therapy remains controversial but can be helpful in patients who still have severe disease despite maximum bronchodilator therapy. Antibiotics can be of benefit in COPD patients undergoing an exacerbation who have increasing dyspnea, cough, and phlegm production.


Foot wounds in diabetic patients. A comprehensive approach incorporating use of topical growth factors.

Managing nonhealing foot wounds in diabetic patients requires an understanding of the wounds' multiple contributing causes, including neuropathy, vascular occlusive disease, infection, and impaired wound healing. Proper attention to each cause may require consultations with vascular or orthopedic surgeons, diabetic education nurses, podiatrists, orthotists, and pedorthists. Wounds that fail to heal may respond to topical application of growth factors as part of a comprehensive clinical approach to the diabetic foot wound. An aggressive approach to diagnosis and treatment can result in improved wound healing and limb salvage.


[Changes in plasma lipoproteins in Mediterranean boutonneuse fever].

We report 25 cases of boutonneuse fever with, in the acute phase, an increase of plasma transaminases, triglycerides and apoprotein B levels, a decrease of total, HDL and LDL cholesterol and a decrease of apoprotein A. These changes disappeared 1 to 4 weeks after the beginning of treatment. They might be due to a reduced activity of lecithin-cholesterol acetyltransferase and lipoprotein lipase, probably caused by the hepatic and vascular disorders frequently found in this disease.


Time-dependent alterations of leukotriene production and catabolism in rat peritoneal macrophages following intraperitoneal injection of thioglycollate broth.

Alterations of leukotriene (LT) productivity in peritoneal macrophages (PM) from untreated rats (control) as well as from rats treated i.p. with thioglycollate broth (TG) were investigated on days 3, 7 and 14 after TG administration. The resident PM from the untreated rats produced mainly LTB4 and 5-HETE with small amounts of 12-HETE and LTD4 with only a trace of LTC4 when stimulated with the calcium ionophore A23187. The PM elicited from rats on days 3 and 7 produced more LTC4 than did the resident PM but fewer other lipoxygenase metabolites. On day 14, however, the elicited PM resembled the resident PM in terms of lipoxygenase metabolite production. Similar results were achieved in the presence of arachidonic acid and A23187. A decrease in lipoxygenase metabolism in the elicited PM was also suggested by using opsonized zymosan. Catabolism studies indicated a reduction in r-glutamyl transpeptidase activity in the elicited PM and suggested a reduction in catabolism for LTB4 in the former cells. The authors conclude that the TG-elicited PM generate fewer lipoxygenase metabolites than the resident PM following stimulation, but show a preferential conversion of LTA4 to sulfidopeptide LTs rather than to LTB4. The elicited PM also show a reduced catabolism for LTC4 and LTB4.


Efficacy of moclobemide in different patient groups: a meta-analysis of studies.

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