Evidence of a natural killer (NK) cell repertoire for (allo) antigen recognition: definition of five distinct NK-determined allospecificities in humans.

Previous studies indicated that CD3-CD16+ natural killer (NK) cells are capable of specific alloantigen recognition. Thus, alloreactive NK clones lysed normal allogeneic target cells (phytohemagglutinin [PHA] blasts) bearing the stimulating alloantigen but did not lyse autologous cells or the majority of unrelated allogeneic cells. In this study we investigated whether NK cells isolated from single individuals could exhibit different allospecificities. To this end, we derived large numbers of CD3-CD16+ clones (in the presence of PHA) from fresh CD3- peripheral blood lymphocytes. Cloning efficiencies ranged between 5 and 10%. The resulting CD3-CD16+ clones were tested for their reactivity against a panel of allogeneic PHA blasts (derived from six donors). In a given individual (A), four distinct groups of clones could be identified according to their pattern of reactivity (over 400 clones have been analyzed). Clones that could be assigned to one or another group of specificity represented 36% of all clones derived from this donor. The remaining clones did not display cytolytic activity against any of the allogeneic target cells used in the panel. None of the clones lysed autologous (A) PHA blasts, yet, these cells were lysed by the representative clones G10 and H12 specific for donor A. Clones displaying a cytolytic pattern of reactivity identical to that defined for donor A were present in other individuals studied, however not all groups of allospecific clones were necessarily represented in different individuals. Allospecific clones belonging to the various groups were homogeneous in the expression of EB6/GL183-triggering surface molecules, and could thus be assigned to one or another of the previously defined subsets of NK cells. Genetic analysis of the new NK-defined alloantigens was performed in representative families. The corresponding characters were found to segregate independently and, at least for three of them, an autosomic recessive type of inheritance could be demonstrated. Moreover, the comparative analysis of the segregation of the major histocompatibility complex haplotypes and the recessive or dominant alleles of the genes governing the five specificities analyzed indicated that there is no independent sampling between the two genetic traits, thus suggesting that the genes regulating the NK-defined specificities are carried by chromosome 6. Finally, some donors expressed more than one specificity, thus providing evidence for an NK-defined complex haplotype.


Growth factor-dependent inhibition of normal hematopoiesis by N-ras antisense oligodeoxynucleotides.

To determine whether N-ras expression is required at specific stages of the process of in vitro normal human hematopoiesis, adherent- and T lymphocyte-depleted mononuclear marrow cells (A-T-MNC) or highly purified progenitors (CD34+ cells) were cultured in semisolid medium, under conditions that favor the growth of specific progenitor cell types, after exposure to N-ras sense and antisense oligodeoxynucleotides. N-ras antisense, but not sense, oligodeoxynucleotide treatment of A-T-MNC and CD34+ cells resulted in a significantly decreased number of granulocyte/macrophage colony-forming units (CFU-GM) induced by interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-CSF) and of macrophage colonies (CFU-M) induced by M-CSF, but not of granulocytic colonies induced with G-CSF or IL-5. However, the same treatment significantly inhibited colony formation induced by each of the above factors in combination with IL-3. Megakaryocytic colony (CFU-Meg) formation from A-T-MNC or CD34+ cells in the presence of IL-6 + IL-3 + erythropoietin (Epo) was also markedly decreased after antisense oligodeoxynucleotide treatment. Erythroid colonies derived from A-T-MNC in the presence of Epo (CFU-E) were not inhibited upon antisense treatment, whereas those arising from A-T-MNC or CD34+ cells in the presence of IL-3 + Epo (BFU-E) were markedly affected. These results are consistent with the hypothesis that distinct signal transduction pathways, involving N-ras or not, are activated by different growth factors in different hematopoietic progenitor cells.


Preferential V beta gene usage and lack of junctional sequence conservation among human T cell receptors specific for a tetanus toxin-derived peptide: evidence for a dominant role of a germline-encoded V region in antigen/major histocompatibility complex recognition.

To investigate the structural and genetic basis of the T cell response to defined peptide/major histocompatibility (MHC) class II complexes in humans, we established a large panel of T cell clones (61) from donors of different HLA-DR haplotypes and reactive with a tetanus toxin-derived peptide (tt830-844) recognized in association with most DR molecules (universal peptide). By using a bacterial enterotoxin-based proliferation assay and cDNA sequencing, we found preferential use of a particular V beta region gene segment, V beta 2.1, in three of the individuals studied (64%, n = 58), irrespective of whether the peptide was presented by the DR6wcI, DR4w4, or DRw11.1 and DRw11.2 alleles, demonstrating that shared MHC class II antigens are not required for shared V beta gene use by T cell receptors (TCRs) specific for this peptide. V alpha gene use was more heterogeneous, with at least seven different V alpha segments derived from five distinct families encoding alpha chains able to pair with V beta 2.1 chains to form a tt830-844/DR-specific binding site. Several cases were found of clones restricted to different DR alleles that expressed identical V beta and (or very closely related) V alpha gene segments and that differed only in their junctional sequences. Thus, changes in the putative complementary determining region 3 (CDR3) of the TCR may, in certain cases, alter MHC specificity and maintain peptide reactivity. Finally, in contrast to what has been observed in other defined peptide/MHC systems, a striking heterogeneity was found in the junctional regions of both alpha and beta chains, even for TCRs with identical V alpha and/or V beta gene segments and the same restriction. Among 14 anti-tt830-844 clones using the V beta 2.1 gene segment, 14 unique V beta-D-J beta junctions were found, with no evident conservation in length and/or amino acid composition. One interpretation for this apparent lack of coselection of specific junctional sequences in the context of a common V element, V beta 2.1, is that this V region plays a dominant role in the recognition of the tt830-844/DR complex.


Anchoring pockets in human histocompatibility complex leukocyte antigen (HLA) class I molecules: analysis of the conserved B ("45") pocket of HLA-B27.

Dissection of the peptide binding grooves of seven subtypes of human histocompatibility leukocyte antigen (HLA)-B27 into the six specificity pockets defined by the 2.6-A structure of HLA-A*0201 revealed just one pocket, the B ("45") pocket, that is conserved among all the HLA-B27 subtypes. Functional studies of mutant HLA-B*2705 molecules with point substitutions in residues of the B pocket show that this structure, and the glutamine residue at position 45 in particular, plays a critical role in cell surface expression, peptide binding, and in the presentation of both exogenous and endogenous peptides by HLA-B*2705. We predict that the B pocket of HLA-B*2705 interacts with an amino acid side chain that anchors peptides in the binding groove, and that this peptide motif is present in most endogenously processed peptides that bind to all seven subtypes of HLA-B27.


Alloreactive cytotoxic T lymphocytes recognize epitopes determined by both the alpha helices and beta sheets of the class I peptide binding site.

A chimeric class I glycoprotein was created to investigate the functional contribution of the alpha helices and the beta-pleated sheets in forming the antigen recognition site (ARS) of antigen-presenting molecules. This novel molecule was generated by replacing the DNA sequences encoding the alpha helices of the Ld gene with the corresponding sequences from the Kb gene. Serologic analysis of transfected L cells that expressed the chimeric molecule (Kb alpha Ld beta) revealed that the engineered class I glycoprotein retains two conformational epitopes associated with the alpha helices of Kb, as defined by monoclonal antibodies K10.56 and 28-13-3. These results demonstrate that the alpha helices of Kb can associate with the beta-pleated sheets of Ld to form a stable structure, which is expressed on the cell surface. To address the role of the alpha helices of the ARS in determining T cell crossreactivity, alloreactive cytotoxic T lymphocytes (CTL) were used to analyze L cells expressing Kb alpha Ld beta. CTL raised against Kb or Ld as alloantigens showed little, if any, ability to lyse L cells expressing Kb alpha Ld beta. Thus, alloreactive CTL did not recognize structures determined by the alpha helices alone or by the beta sheets of the ARS alone. However, bulk and cloned alloreactive CTL that were generated against the mutant Kb glycoprotein Kbm8 reacted strongly with Kb alpha Ld beta. In addition to the Kb alpha helices, the Kbm8 ARS shares a single polymorphic amino acid at position 24 with Kb alpha Ld beta. Amino acid 24 is located on the beta 2 strand that forms part of the floor of the ARS and has been identified as a component of pocket B in the HLA class I ARS. The substitution of Glu to Ser at this position was shown previously to be the central determinant of the Kbm8 mutant alloantigenicity. The functional significance of this position in determining crossreactivity between bm8 and Kb alpha Ld beta identifies pocket B as a strong anchor for allogenic self-peptides. These findings demonstrate that determinants recognized by CTL on class I alloantigens are formed by interactions involving both the alpha helices and beta sheets of the ARS. These interactions are best explained by the influence of the alpha helices and beta sheets on the peptide-binding properties of these antigen-presenting molecules.


Clonal deletion of postthymic T cells: veto cells kill precursor cytotoxic T lymphocytes.

Veto cell-mediated suppression of cytotoxic T lymphocyte (CTL) responses has been proposed as one mechanism by which self-tolerance is maintained in mature T cell populations. We have previously reported that murine bone marrow cells cultured in the presence of high-dose interleukin 2 (IL-2) (activated bone marrow cells [ABM]) mediate strong veto suppressor function. To examine mechanisms by which ABM may suppress precursor CTL (p-CTL) responses, we used p-CTL generated from spleen cells of transgenic mice expressing a T cell receptor specific for H-2 Ld. It was demonstrated that the cytotoxic response by these p-CTL after stimulation with irradiated H-2d/k spleen cells was suppressed by DBA/2 (H-2d) ABM, but not by B10.BR (H-2k) ABM or dm1 (Dd, Ld mutant) ABM. Flow cytometry analysis with propidium iodide staining revealed that these p-CTL were specifically deleted by incubation with H-2d ABM, but not with H-2k ABM. These data indicate that ABM veto cells kill p-CTL with specificity for antigens expressed on the surface of the ABM, and that the mechanism for veto cell activity of ABM is clonal deletion of p-CTL.


Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts.

Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady-state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.


Ca(2+)-activated K+ channels in human leukemic T cells.

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.


Sustained and transient calcium currents in horizontal cells of the white bass retina.

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch-clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15-60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent.


Distribution of the blood-brain barrier in heterotopic brain transplants and its relationship to the lesions of EAE.

The blood-brain barrier (BBB) is recognized as a barrier to the trafficking of molecules and cellular elements into the central nervous system (CNS). Horseradish peroxidase (HRP) exclusion is used as a measure of BBB integrity. The BBB is altered and becomes permeable during the course of experimental allergic encephalomyelitis (EAE). Heterotopic brain transplantation into the anterior eye chamber is a technique for studying genetic influences and the role of individual cell types on the development of EAE. Prior to EAE induction, HRP is excluded from the central portion of the transplant, demonstrating an intact BBB. In contrast, HRP localization is found at the periphery of the transplant, suggesting an incomplete barrier. However, EAE lesions typically occur within the more central regions of the transplant, where the BBB is intact, and not at peripherally located "leaky" areas. This suggests that endothelial cells at intact BBB sites may direct trafficking of lymphocytes (gating) into the CNS during the development of EAE, rather than the passive entry of lymphocytes into the CNS through a leaky BBB.


Regional differentiation of the human fetal ependyma: immunocytochemical markers.

The development of the ependyma from 6 weeks (wk) gestation to term was studied in 26 human fetuses and infants for immunocytochemical differentiation using antibodies against vimentin, several cytokeratins, glial fibrillary acidic protein (GFAP) and S-100 protein. Acridine orange-RNA fluorescence was uniform in all differentiated ependymal cells. Marked differences were demonstrated among various anticytokeratin antibodies. Vimentin was demonstrated in undifferentiated cells, particularly during mitosis, and persisted as the ependyma matured. It was strong in floor plate cells and processes forming the ventral median septum. Vimentin and cytokeratin CK-904 coexisted with other immunoreactive proteins but disappeared in a caudorostral gradient with maturation. At 8 wk gestation, GFAP was detected in roof plate cells and their processes forming the dorsal median septum. S-100 protein appeared as early as 6 wk and had a more restricted regional distribution than GFAP at all ages. It was strong in the basal plate ependyma of the spinal cord in young fetuses. The temporal and spatial distributions of the immunoreactive proteins studied correlate with evidence that fetal ependymal cells synthesize compounds that attract or repel axonal growth cones to prevent axons from entering the ventricles or deviating from programmed projection pathways. An additional role may be to induce the transformation of radial glial cells in the subventricular zone.


Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals.

Living motor nerve terminals from several species can be stained in an activity-dependent fashion by certain styryl dyes, such as RH414, RH795, and a new dye, FM1-43, which can be imaged independently of the others. The dyes evidently become trapped within recycled synaptic vesicles. In frog cutaneus pectoris muscle, bright fluorescent spots spaced regularly along the length of the nerve terminals appear after stimulation in the presence of the dye. The spots align well with postsynaptic ACh receptors and are persistent for many hours, unless further stimulation is given, in which case the spots disappear. Destaining, like staining, requires transmitter release and proceeds gradually over several minutes at high stimulus frequencies (e.g., 30 Hz), and fluorescent spots in the same terminal disappear at about the same rate. We suggest that each spot is a cluster of hundred of synaptic vesicles and that the mechanism of staining involves the ability of the dyes to partition reversibly into the outer leaflet of surface membranes, without being able to penetrate the entire membrane thickness. Then, during endocytosis following transmitter release, dye molecules become trapped in recycled synaptic vesicle membranes. The dyes therefore make it possible optically to study vesicle exocytosis and recycling in living nerve terminals in real time, and should be useful for marking terminals in a variety of preparations according to their level of activity.


Axonogenesis and morphogenesis in the embryonic zebrafish brain.

We have examined early neuronal differentiation and axonogenesis in the fore- and midbrain of zebrafish embryos to address general issues of early vertebrate brain development. AChE expression and HNK-1 antibody immunoreactivity were used as markers for differentiated neurons and axons, respectively. The pattern of neuronal differentiation followed a stereotyped sequence. AChE-positive cells first appeared between 14 and 16 hr in three small, isolated, bilaterally symmetrical clusters on the surface of the brain. The three clusters--the dorsorostral, ventrorostral, and ventrocaudal clusters--proved to be the progenitors of the telencephalon, ventral diencephalon, and mesencephalic tegmentum, respectively. With further development, more cells were added to these three clusters, and new clusters appeared in the anlage of the epiphysis (18 hr) and in the pituitary and dorsal mesencephalon (by 24 hr). Subsequently, as more neurons differentiated, the gaps of unlabeled cells were reduced; by 48 hr, the cluster boundaries were indistinguishable. Axonogenesis also followed a stereotyped sequence. The first HNK-1-labeled processes arose from the first three clusters of AChE-positive cells and connected the clusters. The earliest axonal growth cones appeared at 16 hr, directed caudally from two to three neurons of the ventrocaudal cluster and pioneering the ventral longitudinal tract. By 18 hr, the tract of the postoptic commissure was initiated by growth cones directed caudally from the ventrorostral cluster toward the ventrocaudal cluster. By 20 hr, axons from the dorsorostral cluster projected ventrally to form the supraoptic tract. The other dorsoventral tracts (the dorsoventral diencephalic tract and the tract of the posterior commissure) became evident between 20 and 24 hr. These observations provide a continuous record of the topological distortions involved in the conversion of the tubular embryonic brain into the contorted adult form. The telencephalon, ventral diencephalon, and hypothalamus originate from the same rostrocaudal level of the neural tube. The pattern of differentiation demonstrated that the early development of the rostral neural tube occurs simultaneously in several independent centers, similar to the overtly segmental development of the hindbrain.


Development of axonal arbors of layer 4 spiny neurons in cat striate cortex.

Spiny neurons in layer 4 of cat striate cortex are the primary recipients of geniculocortical afferents and provide crucial links to other cortical layers for processing visual information. Using intracellular staining, we examined the development of the local axonal projections of these neurons to determine (1) whether the laminar specificity of their projections emerged specifically or was sculpted from transient exuberant projections and (2) whether the emergence of excitatory connections from layer 4 to layer 2/3 could contribute to the activity-dependent development of clustered horizontal connections of layer 2/3 pyramidal neurons. Differences in the extent of projections to infragranular (layers 5 and 6, which receive sparse projections) versus superficial layers (layers 2/3 and 4, which receive extensive projections) developed specifically from the outset. By postnatal day 15 (P15) projections to infragranular layers matured and were indistinguishable from those in the oldest animal studied (P33). In contrast, projections to superficial layers continued to increase in complexity after P15. Projections within layer 4, which were the most elaborate at all ages studied, reached maturity at about P20, while those to layer 2/3 continued to increase in complexity through P33. No evidence for exuberant projections to any of these cortical layers was observed. At very early postnatal ages (P5) projections to the subplate region were evident. These disappeared by P8-P11, suggesting the presence of transient connections from layer 4 spiny neurons to subplate neurons. Binocular deprivation did not prevent the emergence of projections from layer 4 spiny neurons into layer 2/3 or development of normal laminar differences in projection density. Connections from layer 4 to layer 2/3 emerged after horizontal connections in layer 2/3 were crudely clustered, but in synchrony with the later refinement of clusters. Collaterals from layer 4 cells first crossed into layer 3 at P11, but were extremely short (extending only 50-200 microns beyond the laminar boundary) and uncommon (only 4 of 19 cells). Since by P8 horizontal projections of layer 2/3 pyramidal neurons are already crudely clustered, the emergence of crude clustering is probably independent of layer 4 to layer 2/3 excitatory projections. The proportion of cells projecting to layer 2/3 and the complexity of their arbors both increased in the subsequent weeks, closely matching the timing of both the refinement of crudely clustered horizontal connections and the emergence of visual responsiveness in layer 2/3.(ABSTRACT TRUNCATED AT 400 WORDS)


Activation and desensitization of AMPA/kainate receptors by novel derivatives of willardiine.

Willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione] is a naturally occurring heterocyclic excitatory amino acid present in the seeds of Acacia and Mimosa. A series of 5-substituted willardiines were synthesized in single enantiomeric forms and tested for activity at AMPA/kainate receptors, using whole-cell recording from mouse embryonic hippocampal neurons. The (S)- but not (R)-isomers of willardiine and 5-bromowillardiine were potent agonists, producing rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine (EC50, 1.5 microM) was seven times more potent than (R,S)-AMPA (EC50, 11 microM) and 30 times more potent than willardiine (EC50, 45 microM); the potency sequence was fluoro greater than nitro greater than chloro approximately bromo greater than iodo greater than willardiine. Willardiines produce strikingly different degrees of desensitization: at saturating doses the equilibrium response to the weakly desensitizing agonist (S)-5-iodowillardiine was similar in amplitude to the response to kainate and 10 times larger than the response to the strongly desensitizing agonist (S)-willardiine. The desensitization sequence was fluoro greater than willardiine greater than nitro approximately chloro greater than bromo greater than iodo greater than kainate. Cross-desensitization experiments confirm that willardiines bind to the same receptors activated by kainate and AMPA, and show that both the rapidly desensitizing and equilibrium responses to willardiines are mediated by the same receptor: (S)-5-iodowillardiine blocked activation of the rapidly desensitizing response evoked by (S)-willardiine and (S)-5-fluorowillardiine, while the latter agonists blocked the equilibrium response to (S)-5-iodowillardiine. A slowly decaying inward tail current was recorded after a brief application of (S)-5-fluorowillardiine but not (S)-willardiine, consistent with a model in which willardiines bind with different affinity to desensitized receptors, such that following removal of agonist, receptors trapped in the desensitized state can return to the open state before dissociation of agonist terminates receptor activation. Willardiines are the first compounds characterized in which simple changes in molecular structure are associated with marked differences in the ability of agonists to produce desensitization of AMPA/kainate receptors.


Synaptic plasticity in Drosophila memory and hyperexcitable mutants: role of cAMP cascade.

Activity-dependent synaptic plasticity has been implicated in the refinement and modification of neural circuits during development and learning. Previous studies show that activity-induced facilitation and potentiation are disrupted at larval neuromuscular junctions in the memory mutants dunce (dnc) and rutabaga (rut) of Drosophila. The diminished learning-memory capacity and synaptic transmission plasticity have been associated with altered cAMP levels since dnc affects the cAMP-specific phosphodiesterase and rut affects adenylate cyclase. In this study, the morphology of larval motor axon terminals was examined by anti-HRP immunohistochemistry. It was found that the numbers of terminal varicosities and branches were increased in dnc mutants, which have elevated cAMP concentrations. Such increase was suppressed in dnc rut double mutants by rut mutations, which reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in double-mutant combinations of ether à go-go (eag) and Shaker (Sh) alleles, which display greatly enhanced nerve activity as a result of reduction in different K+ currents. Therefore, we examined combinations of dnc and rut with eag and Sh mutations to explore the possible relation between activity- and cAMP-induced morphological changes. We found that the expanded projections in dnc were further enhanced in double mutants of dnc with either eag or Sh, an effect that could again be suppressed by rut. The results provide evidence for altered plasticity of synaptic morphology in memory mutants dnc and rut and suggest a role of cAMP cascade in mediating activity-dependent synaptic plasticity.


Transmitter-operated channels in rabbit retinal astrocytes studied in situ by whole-cell patch clamping.

Glutamate and GABA open ion channels in the membranes of astrocytes found on the vitreal surface of the rabbit retinal visual streak. The glutamate-operated channels are opened by kainate, quisqualate, and AMPA, but not by NMDA, aspartate, or the metabotropic agonist 1-aminocyclopentane-1 S,3R-dicarboxylic acid. The effects of glutamate and its analogs can be blocked by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione. The conductance increase evoked by 10 microM glutamate, a concentration of this transmitter near to that found in the vitreous humor that bathes these cells, was equivalent to 22% of the cell's resting conductance. The conductance increase evoked by 1 microM GABA, a concentration near that found in the vitreous, was equivalent to 131% of the cell's resting conductance. The effects of GABA can be blocked by bicuculline. These data show that GABA, and non-NMDA-type glutamate receptors play an important part in determining the resting potential of visual streak astrocytes in situ and that these channels may be of general importance for the functions of astrocytes in vivo.


The effects of pulsed electromagnetic fields on blood vessel growth in the rabbit ear chamber.

A double-blind, controlled trial of the effects of pulsed electromagnetic fields on capillary growth in the rabbit ear chamber in adult New Zealand white rabbits has been performed. Three waveforms have been investigated. The first, a pulse burst waveform, produced a significant increase in the rate of growth of the vascular tissue within the chamber, but had no effect on macroscopic tissue maturation. The second and third, two different single pulse waveforms, had, in contrast, no significant effect on the rate of vascular growth and only an effect on vessel characteristics, with increased maturation of vessels using the second waveform. It is concluded that some of the observed effects of pulsed electromagnetic fields on tissue healing may be mediated through a primary effect on vascular growth.


Type- and group-specific continuous antigenic determinants of HTLV. Use of synthetic peptides for serotyping of HTLV-I and -II infection.

The human T lymphotropic viruses (HTLV-I and -II) are relatively common in subpopulations of certain countries, notably intravenous drug abusers in North America. Infections with these malignancy-associated human retroviruses are hard to discriminate with currently available commercial serological tests. We studied the distribution of antigenicity and the degree of cross-reactivity of epitopes in gag and env of the two viruses. Sequences in the carboxyl terminus of the matrix protein (MA) and the middle of the outer glycoprotein (SU) reacted in a type-specific fashion, while sequences from the capsid protein (CA), the carboxyl terminus of SU, and conserved portions of the transmembrane protein (TM) mainly reacted in a group-specific fashion, correlating with the degree of sequence dissimilarity between the two viruses. The serological discrimination obtained with the peptides was evaluated in a panel of 25 sera where infection with HTLV-I or -II had been typed by competition in a p24 enzyme-linked immunosorbent assay (ELISA) or by the polymerase chain reaction (PCR). After processing peptide results in a computer program, a typing result concordant with earlier results was obtained in 21 of 25 sera. Of the remaining five sera, four were labeled "too weak for typing" and one "HTLV of uncertain type" by the program. They did not react sufficiently strongly or clearly with the peptides to allow classification. A combination of synthetic peptides may become useful for serotyping HTLV infection and become an alternative to Western blots for confirmation of HTLV positivity.


Isolation of haemin-binding proteins of Neisseria gonorrhoeae.

Although Neisseria gonorrhoeae can use haem as the sole exogenous iron source for growth in vitro, the mechanism of haem-iron uptake in the gonococcus is unknown. Two haemin-binding proteins (HmBPs) of 97 and 44 Kda were isolated by batch ligand affinity-chromatography from whole cells or total membranes of gonococci grown under iron-limited conditions but not from those grown under iron-sufficient conditions. Competition binding experiments indicated that the haemin-protein interaction was specific; only haemin or haem-containing proteins, such as human haemoglobin or equine cytochrome c111, but not protoporphyrin IX, iron loaded human transferrin or lactoferrin, could abrogate binding. Identical HmBPs were isolated from three other clinical gonococcal strains, suggesting that these may be interstrain structural and functional homogeneity amongst these polypeptides.


The effects of burn injury on the acute phase response.

The time course of changes in the levels of acute-phase-reactant (APR) mRNAs in different tissues of rats with a 10% or a 60% total-body-surface-area (TBSA) burn and the relationship between the induction of APRs and the host's tolerance to thermal injury were studied. The acute phase response in a LPS-induced inflammation model and a burn-plus-LPS model were compared. The results of this study indicated that (1) the major site of APR synthesis is the liver; (2) even a small surface burn injury can elicit a rapid acute phase response, but the intensity of APR expression increases with the severity of the burn; (3) the down regulation of albumin mRNA, which is characteristic of the acute phase response, does not occur even though transferrin (Trf) mRNA levels are significantly decreased; (4) the resistant strain of inbred rats showed higher levels of alpha 1-antitrypsin (AT) mRNA before and after burn injury, indicating its contribution to the host's tolerance to thermal injury; (5) the increases in alpha 1-acid glycoprotein (AGP) and AT expressions are limited in the burn-plus-LPS rat model compared with either the burn model or LPS-stimulated model alone.


Endogenous interferon and triglyceride concentrations to assess response to zidovudine in AIDS and advanced AIDS-related complex.

To improve evaluation of new antiretroviral drugs in the acquired immunodeficiency syndrome (AIDS), sensitive biological markers that accurately predict response to treatment are needed. Two possible markers are endogenous interferon (E-IFN), which is a cytokine involved in the pathophysiology of AIDS, and serum triglycerides (TG), which are raised in patients with AIDS, possibly reflecting enhanced cytokine activity. E-IFN, TG, body-mass index, CD4 count, and HIV p24 were measured in 19 patients (15 with AIDS, 4 with AIDS-related complex), who were part of the phase II licensing trial of zidovudine (ZDV). 10 received ZDV and 9 received placebo. Rapid, significant, and sustained declines from initial values in E-IFN and TG concentrations were observed in ZDV patients but not in placebo patients. Baseline values of E-IFN and TG concentrations after 4 months on ZDV treatment were both important contributors to long-term survival. The findings suggest that these indicators of abnormal cytokine expression may be useful measures of not only disease severity but also efficacy of antiretroviral therapy in AIDS.


Failure to detect cytomegalovirus DNA in pancreas in type 2 diabetes.

We have attempted to confirm the finding of cytomegalovirus (CMV) nucleic acid sequences in pancreas sections from patients with type 2 diabetes in a large population since this finding has major implications for the pathogenesis of the disorder. A highly sensitive nested polymerase chain reaction method was developed to detect the immediate-early CMV gene in DNA extracted from wax-embedded tissue sections. We could not confirm the previous findings; CMV DNA was not detected in pancreas sections from 43 type 2 diabetic patients or from 38 non-diabetic age-matched subjects, although the method detected CMV DNA, even in very low concentrations, in positive controls.


The effect of some new platinum (II) and palladium (II) coordination complexes on rat hepatic nuclear transcription in vitro.

Several new L-amino acid derivatives of 2,2'-bipyridine and 1,10-phenanthroline complexes of platinum (Pt) and palladium (Pd) and a few binuclear 2,2'-bipyridine complexes of these metals were tested for their potential to inhibit rat hepatic nuclear transcription in vitro. Pd complexes were generally more effective inhibitors of transcription than the corresponding Pt complexes. Among Pd-diimine chlorides, the 2,2'-bipyridine complex was nearly 10 times more active than the corresponding 1,10-phenanthroline complex. Both Pt-diimine chlorides, however, showed same level of inhibitory activity. Amino acid derivatives were less inhibitory with respect to the parent metal diimine chlorides except for 1,10-phenanthroline complexes of Pd. For binuclear 2,2'-bipyridine complexes of Pt, the increase in length of linking hydrocarcon chain increased the inhibitory potential of the complex. The mechanism of inhibition of transcription by these metal complexes was sought to be understood by use of actinomycin-D and poly[d(I-C)] to differentiate effect on the two major components of transcription machinery viz. the template and the enzyme. These studies along with studies on reconstituted system of transcription using either pretreated template or enzyme indicate that these metal complexes displayed dual effect on transcription by inhibiting both the template and the enzymes.


Cloning of a 21.7-kDa vaccine-dominant antigen gene of Schistosoma mansoni reveals an EF hand-like motif.

Several cDNA clones encoding a 21.7-kDa antigen (Sm21.7) were detected from a Schistosoma mansoni sporocyst cDNA expression library using irradiated cercaria-vaccinated rabbit serum. The antigen was designated 'vaccine dominant' because parasite-derived Sm21.7 was recognised preferentially by mouse vaccine sera compared with mouse infection sera. The cDNA and corresponding gDNA sequences showed 64% identity at the nucleotide level and 47% identity at the amino acid level with the sequence of a previously described S. mansoni tegumental antigen, sma22.6. Whereas sma22.6 mRNA occurs almost exclusively in the adult worm, Sm21.7 mRNA was equally abundant in the sporocyst, schistosomular and adult stages. Both Sm21.7 and sma22.6 sequences reveal a motif strongly homologous to the EF hand calcium binding domain but lacking the invariant glycine in the calcium binding loop. The disruptive nature of the glutamine which in Sm21.7 replaces the glycine explains why the motif is non-functional, as shown by the inability of Sm21.7 to bind calcium.


Some kinetic properties of pyruvate kinase from Trypanosoma brucei.

We have studied the kinetics of the allosteric interactions of pyruvate kinase from Trypanosoma brucei. The kinetics for phosphoenolpyruvate depended strongly on the nature of the bivalent metal ions. Pyruvate kinase activated by Mg2+ had the highest catalytic activity, but also the highest S0.5 for phosphoenolpyruvate, while the opposite was true for pyruvate kinase activated by Mn2+. The reaction rates of Mg(2+)-pyruvate kinase and Mn(2+)-pyruvate kinase were clearly allosteric with respect to phosphoenolpyruvate, while the kinetics with Co(2+)-pyruvate kinase were hyperbolic. However, Co(2+)-pyruvate kinase was still sensitive to heterotropic activation. Trypanosomal pyruvate kinase is unique in that the best activator was fructose 2,6-bisphosphate. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate were also strong heterotropic activators, which were much more effective than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate. In the presence of the heterotropic activators, the sigmoidal kinetics with respect to phosphoenolpyruvate and the bivalent metal ions were modified as were the concentrations of phosphoenolpyruvate and the bivalent metal ions needed to attain the maximal activity. Maximal activities were not significantly changed with Mg2+ and Mn2+ as the activating metal ions. Moreover, with Co2+ and fructose 2,6-bisphosphate or ribulose 1,5-bisphosphate or 5-phosphorylribose 1-pyrophosphate, the maximal activity was significantly reduced. Ribulose 1,5-bisphosphate and 5-phosphorylribose 1-pyrophosphate resembled fructose 2,6-bisphosphate rather than fructose 1,6-bisphosphate and glucose 1,6-bisphosphate in their action in that the K0.5 values for the former 3 compounds increased when Mg2+ was replaced by Co2+, while the K0.5 for fructose 1,6-bisphosphate and glucose 1,6-bisphosphate increased.(ABSTRACT TRUNCATED AT 250 WORDS)


Structure and expression of the gene for Pv200, a major blood-stage surface antigen of Plasmodium vivax.

Molecular cloning and structure analysis of the gene encoding the Pv200 protein of the Sal-1 strain of Plasmodium vivax revealed an overall identity of 34-37% when the deduced amino acid sequence was compared with the sequences of various major merozoite surface antigens of Plasmodium falciparum, Plasmodium yoelii and Plasmodium chabaudi. When the Sal-1 Pv200 sequence was compared with the corresponding sequence from the Belèm strain of P. vivax, it was found that the two merozoite surface antigens were relatively well conserved with an overall amino acid sequence identity of 81%. A region of 23 repeated glutamine residues, found in the sequence of the Belèm isolate was not found, however, in the Sal-1 sequence. Amino- and carboxy-terminal domains of the Pv200 protein were expressed in the yeast Saccharomyces cerevisiae. Each recombinant protein was shown to react with antibodies in sera from splenectomized Bolivian Saimiri monkeys that had been infected previously with P. vivax, and in human sera from individuals with a history of exposure to vivax malaria. The availability of recombinant DNA-derived Pv200 proteins will now allow a full assessment of their utility in the diagnosis and immunoprophylaxis of the benign tertian malaria associated with P. vivax infection.


Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.


Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm.

RNA polymerase II transcripts, heterogeneous nuclear RNAs (hnRNAs), associate in the nucleus with specific proteins that bind premessenger RNA (hnRNP proteins) and with small nuclear ribonucleoprotein particles (snRNPs). These hnRNA-hnRNP-snRNP complexes assemble on nascent transcripts and hnRNA is processed to mRNA in them. HnRNP proteins have been localized to the nucleoplasm and their functions were presumed to be limited to nuclear events in mRNA biogenesis. It was proposed that an exchange of hnRNP for mRNA-binding proteins accompanies transport of mRNA from the nucleus to the cytoplasm. We show here that several of the abundant hnRNP proteins, including A1, shuttle between the nucleus and the cytoplasm. HnRNP proteins may thus also have cytoplasmic functions. Furthermore, when in the cytoplasm, A1 is bound to mRNA and RNA polymerase II transcription is necessary before it can return to the nucleus. We propose that the cytoplasmic ribonucleoprotein complex of mRNA with hnRNP proteins is the substrate of nuclear-cytoplasmic transport of mRNA.


Limitations of image enhancement for the visually impaired.

Image enhancement as an aid for the visually impaired may improve visibility of TV programs and provide portable visual aid. This paper describes the current techniques for image enhancement and their underlying models. The limitations of the various techniques and of potential methods of implementation are high-lighted. Initial work in this area was based on a linear model. The finite dynamic range available in the video display and contamination of the enhanced image by high spatial frequency noise limited the model's usefulness. I propose a method to address some limitations of the original model that considers the nonlinear response of the visual system and requires enhancement of subthreshold spatial information only. This modification may increase the dynamic range available by decreasing the range previously used by the linear models to enhance visible details. However, for the modified technique to be most effective, the enhancement has to be continuously tuned, based on the patient's visual loss and the spatial frequency content of the displayed images. The implications of these limitations for the potential implementation in TV are discussed. Implementation of an image-enhancing visual aid in a head-mounted, binocular, full-field, virtual vision device may cause substantial difficulties. Patient adaptation may be difficult due to head movement and interaction of the vestibular system response with the head-mounted display. An alternate, bioptic design is proposed in which the display is positioned above or below the line of sight to be examined intermittently, possibly in a freeze-frame mode. Such implementation is also likely to be less expensive, enabling more users access to the device.


Challenges in applying autofocus technology to low vision telescopes.

Autofocus (AF) low vision telescopes offer the potential to increase the acceptance and utilization of such low vision aids (LVA) by the visually impaired. Many patients resist conventional manual focus telescopes for a variety of reasons including appearance, field of view, weight, and utility. The elderly who comprise the significant part of the target population may also resist telescopes due to an avoidance of the technical challenge of its use. Although an AF telescope is technically advanced, it may allow for less manipulation by the wearer and hence enable its more effective application to visual tasks, especially in the near- to mid-range where depths of field narrow and the demands for focusing increase. There are many challenges involved in the application of AF technology to LVA including modification of the focusing range, signal processing for physiologically acceptable performance, and power and weight considerations. A preliminary infrared (IR) AF prototype based upon our recent work with the Ocutech Vision Enhancing System (VES) has been produced. Initial findings are presented which address the requirements of a subsequent version as well as the challenges that will be faced to optimize such a device.


Obstacles encountered in the development of the low vision enhancement system.

The Johns Hopkins Wilmer Eye Institute and the NASA Stennis Space Center are collaborating on the development of a new high technology low vision aid called the Low Vision Enhancement System (LVES). The LVES consists of a binocular head-mounted video display system, video cameras mounted on the head-mounted display, and real-time video image processing in a system package that is battery powered and portable. Through a phased development approach, several generations of the LVES can be made available to the patient in a timely fashion. This paper describes the LVES project with major emphasis on technical problems encountered or anticipated during the development process.


The immediate early gene response to a differentiative stimulus is disrupted by the v-abl and v-ras oncogenes.

The immediate early gene activation in response to a differentiative stimulus was investigated in the hematopoietic progenitor cell line 32DC13(G). A transient and coordinated increase in mRNA levels for c-fos, c-jun, jun-B, TIS-7/PC-4, TIS-8/egr-1, and TIS-11 occurred during the first 2 h of treatment with granulocyte colony stimulating factor (G-CSF), which ultimately induces the 32DC13(G) cells to terminally differentiate into neutrophilic granulocytes. This pattern of mRNA induction was disrupted by v-abl and v-ras, two oncogenes known to interfere with G-CSF-induced differentiation of 32DC13(G) cells. Induction of the mRNAs for c-jun and TIS-7/PC-4 was blocked by the presence of v-abl, whereas v-ras caused constitutive expression of c-fos mRNA and blocked the c-jun, jun-B and TIS-7/PC-4 mRNA response. Release of the differentiation block in the ras-transformed 32DC13(G) cells by co-treatment with retinoic acid and G-CSF partially restored the normal c-fos and c-jun mRNA induction pattern, suggesting that the proper activation of these genes may be important for myeloid differentiation.


The biochemical properties and transforming potential of human Wnt-2 are similar to Wnt-1.

Wnt-2 is a member of the Wnt gene family that includes the proto-oncogene Wnt-1 (formerly Int-1). Although the predicted protein product of the Wnt-2 gene has only 38% amino acid identity with Wnt-1, it exhibits significant conservation of structural properties, including a hydrophobic signal sequence and invariant spacing and conservation of 22 cysteine residues. We have sought to characterize the biological and biochemical properties of Wnt family members and here present a characterization of Wnt-2 protein and a comparison with Wnt-1. We demonstrate, using both CHO and AtT-20 cells transfected with human Wnt-2 cDNA, that Wnt-2 encodes a 33 kDa protein that is modified by N-linked glycosylation to a 35 kDa species. Secreted Wnt-2 protein was detected in the culture medium only after cells were treated with suramin indicating that, like Wnt-1 protein, Wnt-2 is tightly associated with the cell surface. Expression of Wnt-2 cDNA in the mammary epithelial cell line C57 mg results in loss of density-inhibited growth and induces a transformed phenotype in monolayer culture similar to the effects produced by Wnt-1. These results indicate that Wnt-2 shares several biochemical and biological characteristics with Wnt-1 and suggests that other Wnt family members, by virtue of conserved structural features, may also exhibit similar properties.


Variable pattern of jun and fos gene expression in different hematopoietic cell lines during interleukin 3-induced entry into the cell cycle.

Jun (c-jun, jun-B and jun-D) and fos (c-fos, fos-B and fra) proteins dimerize to form the family of AP-1 transcriptional activators. If each dimer exhibits unique transactivating properties, then any phenotypic change should show a characteristic pattern of jun and fos expression. To test this hypothesis we have assessed jun and fos RNA expression after stimulation of the factor-dependent cell lines 32D and FDCP1. These hematopoietic progenitor lines become quiescent in G0/G1 after interleukin 3 (IL-3) deprivation, and upon stimulation synchronously enter the cell cycle. 32D cells respond to IL-3 with rapid induction of jun-B and c-fos, followed by induction of jun-D and fra-1, but no rise in c-jun expression. FDCP1 cells show a very different pattern, with induction of c-jun, jun-D and fra-1. To investigate the response of a single cell line to different physiological stimuli we used a 32D subclone engineered to respond to colony stimulating factor 1 (CSF-1). This subclone showed identical induction of jun and fos after stimulation with either CSF-1 or IL-3. The conservation of response of a single cell line, but the disparate patterns demonstrated by different cells, suggest a fundamental difference in both the regulation and function of the fos/jun complexes in these cells.


Loss of c-kit expression in cultured melanoma cells.

The proto-oncogene c-kit encodes a receptor tyrosine kinase which has been shown to play a key role in melanocyte development. In this report we asked whether the c-kit gene product is also involved in promoting the growth of transformed melanocytes. We found that, while c-Kit protein was readily observed in normal human neonatal and adult melanocytes, the majority of cell lines established from human melanoma samples did not express detectable levels of c-kit mRNA or protein. A similar pattern of differential expression was also observed in normal and transformed murine melanocytes. Our findings raise the possibility that a marked reduction in c-kit gene expression either promotes or is a consequence of transformation in melanocytes.


Interferon production during the course of Mycoplasma pneumoniae infection.

In patients infected with Mycoplasma pneumoniae the development of interferon (IFN) was studied in nasopharyngeal secretions and sera. The production of IFN-gamma by lymphocytes was also investigated in response to M. pneumoniae antigen and mumps virus antigen. IFN-alpha was detected in 25 (61.0%) of 41 nasopharyngeal secretion samples and in 25 (59.5%) of 42 serum samples within 6 days after the onset of illness. IFN-alpha was significantly higher in nasopharyngeal secretions than in sera and a significant correlation was observed between the two. In most of the patients lymphocytes produced a larger amount of IFN-gamma in the convalescent stage than in the acute stage, when lymphocytes were stimulated with M. pneumoniae antigen. In some patients, however, lymphocytes did not produce IFN-gamma during the course of illness. Such lymphocytes, negative for IFN-gamma production in response to M. pneumoniae, produced IFN-gamma after the depletion of macrophages, and readdition of macrophages suppressed the production of IFN-gamma by lymphocytes. When lymphocytes were stimulated with heterogeneous antigen (mumps virus), they produced no IFN or a small amount of IFN in the acute stage of M. pneumoniae infection, and IFN production increased in the convalescent stage. Different mechanisms seem to work for homogeneous and heterogeneous antigens in the suppression of IFN production in M. pneumoniae infection.


Hyperbilirubinemia in low birth weight infants and outcome at 5 years of age.

The collaborative national survey on morbidity and mortality in preterm and small for gestational age infants in the Netherlands enrolled initially 1338 infants born in 1983. The relationship between maximal serum total bilirubin concentration in the neonatal period and neurodevelopmental outcome in the survivors of this cohort was studied. This relationship at the corrected age of 2 years was previously reported. A dose-response relationship between maximal serum total bilirubin concentration and risk of adverse outcome was observed in the 831 surviving children. The present study reassessed the relationship at the age of 5 years in 814 children. There was no significant difference in mean maximal serum total bilirubin concentration between the children with and without a handicap. This was confirmed by logistic regression analysis. After correction for seven suspected confounding factors (gestational age, birth weight, intracranial hemorrhage, ventriculomegaly, seizures, bronchopulmonary dysplasia, and socioeconomic status) the estimated odds ratio was 1.2 (confidence interval 0.89, 1.43) per 50 mumol/L increase of total bilirubin. However, in this analysis an interaction between bilirubin and intracranial hemorrhage was observed. Therefore, the cohort was divided into two groups according to the absence or presence of an intracranial hemorrhage. Logistic regression analysis including four suspected confounding factors (gestational age, ventriculomegaly, seizures, and socioeconomic status) was then again applied. In children who had suffered from an intracranial hemorrhage in the neonatal period the estimated odds ratio was 1.84 (confidence interval 1.08, 3.15) per 50 mumol/L increase of bilirubin. Similar results were obtained treating bilirubin as a categorized exposure. The odds ratio in children without a hemorrhage was 1.05 (confidence interval 0.80, 1.38), probably because of the small number of surviving handicapped children.


Infant Health and Development Program for low birth weight, premature infants: program elements, family participation, and child intelligence.

The Infant Health and Development Program was an eight-site randomized controlled trial testing the efficacy of early intervention to enhance the cognitive, behavioral, and health status of low birth weight, premature infants. The 377 intervention families received for the first 3 years of life: (1) pediatric follow-up, (2) home visits, (3) parent support groups, and (4) a systematic educational program provided in specialized child development centers. The control group (n = 608) received the same pediatric follow-up and referral services only. This paper describes the delivery of the intervention and its outcomes. A Family Participation Index that was the sum of participation frequencies in each of the program modalities unique to the intervention revealed that program implementation was not different across the eight sites. Index scores did not vary systematically with mother's ethnicity, age, or education or with child's birth weight, gender, or neonatal health status; but they were positively related to children's IQ scores at age 3. Only 1.9% of children of families in the highest tercile of participation scored in the mentally retarded range (IQ less than or equal to 70), whereas 3.5% and 13% of children in the middle and lowest participation terciles, respectively, scored in the retarded range. Similar findings were obtained for borderline intellectual functioning. These findings are consistent with previous research linking intensity of intervention services with degree of positive cognitive outcomes for high-risk infants. The determinants of variations in individual family participation remain unknown.


Identification and management of psychosocial and developmental problems in community-based, primary care pediatric practices.

The importance of psychological and social issues for children's well-being has long been recognized and their importance in the practice of pediatrics is well documented. However, many of the studies looking at this issue have emphasized psychiatric problems rather than issues commonly referred to as the new morbidity. The goal of this research was to refocus interest on the problems of the new morbidity. This study examined the rates and predictors of psychological problems in 19 of 23 randomly chosen pediatric practices in the greater New Haven area. Families of all 4- to 8-year-old children were invited to participate and to complete the Child Behavior Checklist prior to seeing a clinician. Clinicians completed a 13-category checklist of psychosocial and developmental problems based on a World Health Organization-sponsored primary care, child-oriented classification system. Of the 2006 eligible families, 1886 (94%) participated. Clinicians identified at least one psychosocial or developmental problems in 515 children (27.3%). Thirty-one percent of the children with problems received no active intervention, 40% received intervention by the clinician, and 16% were referred to specialty services. Not surprisingly, children whose problems were rated as moderate or severe were twice as likely to be referred compared with children with mild problems. Recognition of a problem was related to four characteristics: if the visit was for well child rather than acute care; if the clinician felt he or she knew a child well; if the child was male; and if the child had unmarried parents (all P less than or equal to .05).(ABSTRACT TRUNCATED AT 250 WORDS)


Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding.


Expression of the P-protein of the human hepatitis B virus in a vaccinia virus system and detection of the nucleocapsid-associated P-gene product by radiolabelling at newly introduced phosphorylation sites.

Hepatitis B virus (HBV) contains a particle-associated DNA polymerase/reverse transcriptase activity encoded by the P (pol) open reading frame. Due to its low abundance, the corresponding protein has so far escaped direct detection and structural analysis. As a first step to overcome these difficulties, a series of recombinant vaccinia viruses was constructed and used for the synthesis in human hepatoma cells of both the authentic full length protein and of its functional domains. Pulse chase experiments demonstrated that the P-proteins had very short half lives in striking contrast to the viral core protein expressed in parallel with the same system. No evidence was obtained for a specific proteolytic processing of the P-protein as occurring with retroviral pol gene products. Overexpression of P-protein by recombinant vaccinia viruses was then employed to develop a highly sensitive detection method based on the in vitro phosphorylation of newly introduced target sites for protein kinase A. The usefulness of this method was demonstrated in the analysis of encapsidated P-gene products that were transiently expressed from an appropriately modified HBV genome. The results obtained indicate that the P-protein acts unprocessed, at least during the initial steps of nucleocapsid assembly and reverse transcription, and that a fraction of the P-protein molecules is linked as such to the viral DNA. Direct detection of the hepadnaviral P-protein by in vitro phosphorylation should greatly facilitate future analyses on P-protein structure and function.


Substitutions of a cysteine conserved among DNA cytosine methylases result in a variety of phenotypes.

The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it.


The PARP and VSG genes of Trypanosoma brucei do not resemble RNA polymerase II transcription units in sensitivity to Sarkosyl in nuclear run-on assays.

Addition of the ionic detergent N-lauroylsarcosine (Sarkosyl) affects the efficiency of transcription of genes of the protozoan Trypanosoma brucei in nuclear run-on assays. Transcription of the PARP (procyclin or procyclic acidic repetitive protein), variant cell surface glycoprotein (VSG) and ribosomal RNA (rRNA) genes was resistant or increased after addition of Sarkosyl. In contrast, the transcription of seven protein coding house keeping genes and the mini-exon donor RNA (medRNA) genes was completely abolished by the addition of Sarkosyl, while the transcription of the 5S rRNA genes showed an intermediate sensitivity. We conclude that Sarkosyl can be used to discriminate between the different types of trypanosome transcription units. The PARP and VSG protein coding genes had previously been postulated to be transcribed by an RNA polymerase I-like enzyme on the basis of their resistance to the RNA polymerase II inhibitor alpha-amanitin. This model is now supported by their resistance to the addition of Sarkosyl.


The acceptor stem in pre-tRNAs determines the cleavage specificity of RNase P.

As the result of an unusual RNase P specificity, some special, mature tRNAs have acceptor stems with eight instead of the common seven base pairs. The data from numerous studies suggest that some features in the tRNA domain of pre-tRNAs are important for this behaviour. Here, we show that only five base pairs in the acceptor stem of bacterial histidine tRNAs are required to obtain the changed cleavage site in an unrelated eukaryotic serine tRNA.


The kinetics and specificity of cleavage by RNase P is mainly dependent on the structure of the amino acid acceptor stem.

Cleavage by RNase P of the tRNA(His precursor yields a mature tRNA with an 8 base pair amino acid acceptor stem instead of the usual 7 base pair stem. Here we show, both in vivo and in vitro, that this is mainly dependent on the primary structure and length of the acceptor stem in the precursor. Furthermore, the tRNA(His) precursor used in this study was processed with a change in both kinetic constants, Km and kcat, in comparison to the kinetics of cleavage of the precursor to tRNA(Tyr)Su3. Cleavage of a chimeric tRNA precursor showed that these altered kinetics were due to a difference in the primary structure and in the length of the acceptor stems of these two tRNA precursors. We also studied the cleavage reaction as a function of base substitutions at positions -1 and/or +73 in the precursor to tRNA(His). Our results suggest that the nucleotide at position +73 in tRNA(His) plays a significant role in the kinetics of cleavage of its precursor, possibly in product release. In addition, it appears that the C5 protein of RNase P is involved in the interaction between the enzyme and its substrate in a substrate-dependent manner, as previously suggested.


Analysis of the proximal transcriptional element of the myelin basic protein gene.

The gene encoding myelin basic protein (MBP) contains multiple activator sequences spanning upstream of its transcriptional initiation site which differentially promote transcription in glial cells. The proximal activator sequence, designated MB1, activates transcription in a glial cell type specific manner. This sequence resides between -14 to -50 with respect to the RNA initiation site of the MBP gene. We have identified within the MB1 sequence a 10-nucleotide domain, 5'-ACCTTCAAAG-3', that increases transcription of a test promoter in glial and Schwann cells. This proximal motif functions in both orientations and specifically interacts with a nuclear protein derived from glial cells. Results of in vivo competition experiments indicate that this 10-nucleotide motif positively contributes to the overall transcriptional activity obtained from the entire MBP promoter in glial cells.


Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.


Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.


Neisseria meningitidis encodes an FK506-inhibitable rotamase.

Eukaryotic peptidyl-prolyl cis-trans isomerases (rotamases) fall into two classes, the cyclophilins inhibited by cyclosporin A and the FK506-binding proteins inhibited by the macrolide antibiotic FK506. In prokaryotes homologs of cyclophilins have been identified and found to have rotamase activity. Sequence similarities have been noted between FK506-binding proteins and gene products in a number of bacterial species, but whether these bacterial proteins have rotamase activity is not known. Using the polymerase chain reaction, we have cloned and sequenced a homolog of an FK506-binding protein from Neisseria meningitidis and expressed the gene product as a fusion protein with maltose-binding protein. The fusion protein was purified by affinity chromatography. By measuring the rate of chymotrypsin cleavage of the substrate succinyl-Ala-Ala-Pro-Phe p-nitroanilide, we found that the fusion protein had rotamase activity comparable to that of human FK506-binding protein. This rotamase activity was inhibited by FK506.


Identification and synthesis of a major conserved antigenic epitope of Trypanosoma cruzi.

A gene sequence encoding an immunodominant protein with a repetitive epitope from the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, was cloned and expressed. The identified 10-amino acid repeat is present within a high-molecular-weight trypomastigote antigen that appears specific to and conserved among T. cruzi isolates. More importantly, greater than 95% of T. cruzi infection sera, including both chronic and acute Chagas disease, contained elevated levels of antibody to a 15-amino acid synthetic peptide bearing the repetitive B-cell epitope. Considering the wide diversity of T. cruzi parasites, as well as the broad spectrum of clinical manifestations of Chagas disease, such a prevalent immune response among patients is significant and applicable to the control of Chagas disease through the diagnosis of T. cruzi infection.


Calcium-activated potassium channels mediate prejunctional inhibition of peripheral sensory nerves.

Activation of several receptors, including mu-opioid, alpha 2-adrenergic, and neuropeptide Y receptors, inhibits excitatory nonadrenergic noncholinergic (NANC) neural responses in airways, which were mediated by the release of peptides from capsaicin-sensitive sensory nerves. This raises the possibility of a common inhibitory mechanism, which may be related to an increase in K+ conductance in sensory nerves. To examine this hypothesis, we have studied whether K(+)-channel blockers inhibit the effects of neuromodulators of sensory nerves in guinea pig bronchi by using selective K(+)-channel blockers. Charybdotoxin (ChTX; 10 nM), which blocks large conductance Ca(2+)-activated K(+)-channel function, completely blocked and reversed the inhibitory effects of a mu-opioid agonist, neuropeptide Y, and an alpha 2-adrenoceptor agonist on excitatory NANC responses. Neither inhibitors of ATP-sensitive K+ channels (BRL 31660 or glibenclamide, both at 10 microM) nor an inhibitor of small conductance Ca(2+)-activated K+ channels (apamin; 0.1 microM) were effective. This suggests that ChTX-sensitive K(+)-channel activation may be a common mechanism for the prejunctional modulation of sensory nerves in airways. This may have important implications for the control of neurogenic inflammation.


A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.


The principal neutralization determinant of simian immunodeficiency virus differs from that of human immunodeficiency virus type 1.

To identify the principal neutralization determinant (PND) of simian immunodeficiency virus (SIV), antisera were generated using recombinant gp110 [the SIV analog of the human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein, gp120], gp140, several large recombinant and proteolytic envelope fragments, and synthetic peptides of the SIVmac251 isolate. When purified under conditions that retain its native structure, gp110 bound CD4 and elicited antisera that neutralized SIVmac251 with high titer. Native gp110 also completely inhibited neutralizing antibody in sera from SIVmac251-infected macaques. In contrast, denatured gp110 and gp140, large envelope fragments, and synthetic peptides (including peptides analogous to the HIV-1 PND) elicited very low or undetectable neutralizing antibody titers and did not inhibit neutralizing antibody in infected macaque sera. Enzymatically deglycosylated gp110 efficiently absorbed neutralizing antibodies from macaque sera, showing that neutralizing antibodies primarily bind the protein backbone. A 45-kDa protease digest product, mapping to the carboxyl-terminal third of gp110, also completely absorbed neutralizing antibodies from infected macaque sera. These results show that the PND(s) of this SIV isolate depends on the native conformation and that linear peptides corresponding to the V3 loop of SIV envelope, in contrast to that of HIV-1, do not elicit neutralizing antibody. This may affect the usefulness of SIVmac for evaluating HIV-1 envelope vaccine approaches that rely on eliciting neutralizing antibody.


Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.

The interaction of the mouse c-kit receptor, designated Kit receptor, and steel factor promotes the proliferation and differentiation of hematopoietic progenitor cells. Monoclonal antibodies against the extracellular portion of the mouse Kit receptor were established. Five percent to 10% of total bone marrow cells expressed the Kit receptor, and half of them lack the expression of lineage markers. The Kit receptor was expressed on 70-80% of Thy-1.1lo Lin-Sca-1+ cells, which express Thy-1.1 antigen at a low level and constitute approximately 0.05% of adult bone marrow and fetal liver; by previous studies, these cells have been shown to be highly enriched for multipotent hematopoietic stem cells (HSCs) and are the only hematopoietic cell subset with this activity. Spleen colony formation and long-term multilineage reconstitution activities were contained in the Kit+ but not in the Kit- subpopulations of Thy-1lo Lin-Sca-1+ cells from adult bone marrow, suggesting that the Kit receptor is expressed on HSCs from the earliest stage-i.e., pluripotent HSCs. The role of steel factor in the development and self-renewal of HSCs was tested with Sl/Sl homozygote fetuses, which lack genes to encode functional steel factor. They were shown to have 30-40% of the number of HSCs on days 13-15 when compared with normal litermates. However, the absolute number of HSCs increased during fetal development in the Sl/Sl mice. The results suggest that the Kit receptor-steel factor interaction may not be essential for the initiation of hematopoiesis and the self-renewal of (at least) fetal HSCs.


Macrophage activation and immunomodulation by myeloperoxidase.

Myeloperoxidase (MyPo) is an enzyme found in neutrophils and monocytes that plays an important role in the microbicidal and cytocidal activities of these cells. The present studies show that this enzyme can also affect both capacities and functions of macrophages. When resident peritoneal macrophages from C57BL/6 mice were exposed to preparations of either human or canine enzyme in vitro, tumor necrosis factor (TNF) was released. The amount of TNF produced was dose dependent and could be neutralized with polyclonal anti-TNF. Low levels of interferon were also produced by these cells. In addition, exposure of murine macrophages in vitro to this enzyme resulted in increased ability to destroy 3T12 target cells. Intravenous injection of mice with myeloperoxidase induced the production of both TNF and interferon, which could be detected in the sera. Possible mechanisms of TNF induction include radical production by myeloperoxidase or ligand-receptor interaction by the binding of this enzyme to the mannosyl-fucosyl receptor. These results, when taken in their entirety, suggest that this enzyme can modulate the immune response through effects on macrophage function.


Malignant biliary obstruction: treatment with expandable metallic stents--follow-up of 50 consecutive patients.

A consecutive series of 50 patients with malignant biliary obstruction were treated by means of palliative drainage with a metallic expandable stent. Stent placement was successful in all patients. The patients were followed up prospectively at 2-month intervals over a period of 9-22 months. Forty-one patients (82%) died; nine (18%) are still living. The overall patency and survival rates for the 50 patients were 5.8 months and 7.5 months, respectively. The 30-day mortality rate was 8% (n = 4), the minor complication rate was 18% (n = 9), and the major complication rate was 8% (n = 4). One patient (2%) had intrahepatic arterial bleeding that required embolization, one (2%) had a right subphrenic abscess, and two patients (4%) had transient septic events. Stent occlusion requiring a second intervention occurred in 24% of patients (n = 12). Excellent palliation was achieved in most patients. No stent migration occurred. No great clinical advantages in prolonged patency compared with those of other published series involving the use of plastic stents were demonstrated. Ease of placement and versatility may offset the high cost of the stent.


In vivo degradation of fetal wound hyaluronic acid results in increased fibroplasia, collagen deposition, and neovascularization.

Fetal tissue repair occurs without acute inflammation, prominent fibroplasia, or marked neovascularization. The fetal wound extracellular matrix is rich in hyaluronic acid (HA), while collagen is deposited in an organized normal dermal pattern. In various biologic systems, including regeneration and development, the controlled accumulation and subsequent degradation of hyaluronic acid is associated with distinct cellular and matrix events. Therefore, it is hypothesized that the abundance of hyaluronic acid in fetal wounds may influence cellular and/or matrix events such that tissue repair is highly organized and adult-like scarring does not occur. To test this hypothesis, the hyaluronic acid content of fetal rabbit wounds was reduced by specific degradation with Streptomyces hyaluronidase. Control wounds were treated with either enzyme buffer (n = 12) or denatured enzyme solution (n = 8) and exhibited a normal fetal healing response with scattered peripheral fibroblasts, a matrix of hyaluronic acid, and no infiltrating collagen. In marked contrast, the hyaluronidase-treated wounds (n = 14) demonstrated increased fibroblast infiltration, collagen deposition, and capillary formation. A significant reduction in the hyaluronic acid content of the hyaluronidase-treated wounds was confirmed biochemically. Since the degradation of hyaluronic acid resulted in an altered healing response, this study demonstrates that hyaluronic acid affects the cellular and matrix events in fetal healing and may be partially responsible for the unique qualities of this regenerative repair process.


[Circadian variability of rhythm disorders].

Cardiac arrhythmias exhibit also a circadian variability. It is impressingly apparent in sustained ventricular tachycardia and sudden cardiac death. Adrenergic stimulation during morning hours, a physiologic event for the transition from nocturnal to diurnal activity, appears to be an important arrhythmogenic factor (25). The results of the BHAT-study show that beta blocking agents may substantially reduce the risk for sudden cardiac death during morning hours. This notion should thus be considered in treating patients at risk.


Role of ELAM-1 in adhesion of monocytes to activated human endothelial cells.

The role of ELAM-1 in the adhesion of monocytes to HUVEC, activated for 4h with TNF, was studied using MoAb ENA2 directed against ELAM-1. In a standard adhesion assay at 37 degrees C, F(ab')2 fragments of ENA2 did not, or weakly inhibited adhesion. When metabolic activity of the monocytes was reduced by (i) fixing the monocytes, (ii) performing the adhesion assay at 4 degrees C, and (iii) combining the forementioned conditions, the adhesion of the monocytes was strongly blocked by ENA2 and less effective or not by MoAb IB4 anti-CD18. The pattern of adhesion of monocytes to HUVEC, activated with TNF assessed at 4 degrees C, paralleled ELAM-1 expression on the endothelial cells. Maximal inhibitory effect of ENA2 on adhesion was shown 5 h after activation of HUVEC, at which ELAM-1 expression was also maximal. Adhesion assessed at 37 degrees C remained enhanced for at least 24 h, whereas the inhibitory effect of ENA2 followed ELAM-1 expression. Specific involvement of ELAM-1 was also confirmed using ELAM-1 transfected COS cells. These results indicated that monocytes express a counter structure for ELAM-1 and that this counter structure is involved in adhesion.


Mechanism of transduction by retroviruses.

Retroviruses can capture cellular sequences and express them as oncogenes. Capture has been proposed to be a consequence of the inefficiency of polyadenylation of the viral genome that allows the packaging of cellular sequences flanking the integrated provirus in virions; after transfer into virions, these sequences could be incorporated into the viral genome by illegitimate recombination during reverse transcription. As a test for this hypothesis, a tissue culture system was developed that mimics the transduction process and allows the analysis and quantitation of capture events in a single step. In this model, transduction of sequences adjacent to a provirus depends on the formation of readthrough transcripts and their transmission in virions and leads to various recombinant structures whose formation is independent of sequence similarity at the crossover site. Thus, all events in the transduction process can be attributed to the action of reverse transcriptase on readthrough transcripts without involving deletions of cellular DNA.


Cholinergic and GABAergic mediations of the effects of apomorphine on serotonin neurons.

Apomorphine (APO) has been shown to elevate tryptophan, serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) concentrations in the dorsal raphe (DR) and its corresponding projection site, the striatum, but not in the median raphe (MR) and its terminal area, the hippocampus. We have previously demonstrated that these effects are indirectly mediated through dopamine (DA) autoreceptors in the substantia nigra and possibly gamma-aminobutyric acid (GABA) neurons in or near the DR. In the present study, we have further found that the effects of APO on 5-HT neurons are also mediated through both nicotinic and M1 muscarinic cholinergic receptors as well as GABAA receptors in the DR. This suggestion is based on the findings that both atropine and mecamylamine antagonized the effects of APO, while carbachol at a high dose exerted an effect opposite to that of APO. Besides, pirenzepine and bicuculline at low doses also antagonized, whereas saclofen did not alter the influence of APO on 5-HT in the striatum. Bicuculline at a higher dose enhanced tryptophan and 5-HT measures by itself. None of the drugs studied had a significant effect on tryptophan, 5-HT, or 5-HIAA in the hippocampus. These results together suggest that DA, ACh, and GABA neurons are all involved in the actions of APO on 5-HT, while the direct synaptic relationships among these neurotransmitters and the precise anatomical locus for these interactions to occur are still unknown. It is possible that APO, by inhibiting DA neuron firing in the substantia nigra and through the GABA disinhibition mechanism, therefore indirectly activates 5-HT neurons in the DR and the striatum. While the above neuronal firing model well explains the elevation of 5-HIAA, the simultaneous increases of tryptophan and 5-HT, especially tryptophan, may be more readily explained by a mechanism of tryptophan uptake upon APO administration. Further anatomical, biochemical, and electrophysiological studies are ongoing to test this hypothesis and to clarify the circuit and the anatomical locus (loci) for these interactions to occur.


Differential effects of amfonelic acid on the haloperidol- and clozapine-induced increase in extracellular DOPAC in the nucleus accumbens and the striatum.

This study compares the effects of the nonamphetamine stimulant amfonelic acid on the increase in extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) induced by haloperidol and clozapine in the nucleus accumbens and the striatum of anaesthetized rats. DOPAC was simultaneously recorded in both regions using differential pulse voltammetry with electrically pretreated carbon fibre electrodes. Amfonelic acid (2.5 mg/kg s.c.) did not alter basal striatal DOPAC but produced a significant reduction in extracellular DOPAC in the nucleus accumbens. Haloperidol (1 mg/kg s.c.) increased extracellular DOPAC in both regions. When amfonelic acid was injected 5 min before haloperidol, the increase in DOPAC was potentiated in both the nucleus accumbens and the striatum but with a greater effect in the striatum. Clozapine (30 mg/kg i.p.) increased extracellular DOPAC in both regions, an effect partially attenuated by amfonelic acid in both regions but to a greater extent in the striatum. When ritanserin (5 mg/kg i.p.), a serotonergic antagonist (5-HT-2), was co-administered with haloperidol, the potentiation by amfonelic acid of the increase in extracellular DOPAC induced by haloperidol was attenuated in both the nucleus accumbens and the striatum. The present results confirm that amfonelic acid can be used to discriminate neurochemically between haloperidol and clozapine in vivo. The effects of amfonelic acid on the neuroleptic-induced changes in extracellular DOPAC were greater in the striatum than the nucleus accumbens. These results further demonstrate that both neuroleptics increase dopamine metabolism in the two brain regions but by different mechanisms, supporting the view that the regulation of dopamine metabolism differs in the two regions.(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of the antigenic epitopes of hepatitis B surface antigen involved in the induction of a protective antibody response.

Vaccination with hepatitis B surface antigen (HBsAg) has shown that antibody directed against the common 'a' determinant of this antigen is protective against infection with hepatitis B virus (HBV). In this study the antigenic epitopes of the 'a' determinant have been analysed by competitive inhibition assays and by binding studies to synthetic peptides using a panel of monoclonal antibodies prepared against HBsAg, all of which are shown to recognise the common group determinant. One murine monoclonal antibody used in this study, RFHBs1, has been shown previously to block infectivity of HBV in susceptible chimpanzees ((1983) J. Med. Virol. 16, 89-95). This antibody bound to a cyclical synthetic peptide analogue of amino acids 124 to 137 of the major HBsAg polypeptide.


The immune response to measles virus in mice. T-helper response to the nucleoprotein and mapping of the T-helper epitopes.

The T-helper response to the measles virus nucleoprotein (NP) has been studied in mice. The T-cell proliferative response was measured in lymphocytes from mice immunized with either a vaccinia measles-NP recombinant virus or a mouse neuro-adapted measles virus. A T-cell response was obtained with lymphocytes from H2d or H2k mice when stimulated with either measles virus or the NP expressed in bacteria. The response was CD4+ specific. The T-helper epitopes were mapped using truncated NP peptides. The major epitopes in both H2d and H2k mice were determined to be between amino acids 67-98. A further T-cell epitope (between amino acids 457-525) was identified when H2d mice were immunized with measles virus. Studies to quantitate the precursor cells for these epitopes confirmed that the region 67-98 of NP was immunodominant in both haplotypes immunized with the vaccinia-NP recombinant virus, whereas an additional major epitope was observed in the measles virus-infected H2d mice. The primary structure of the epitopes determined here are compared to predicted T-cell epitope motifs.


Characterization and prognostic significance of silent myocardial ischemia on predischarge electrocardiographic monitoring in unselected patients with myocardial infarction.

Characteristics and prognostic significance of ischemic ST changes at predischarge Holter monitoring were evaluated in 270 consecutive postinfarction patients. The 64 patients with ST changes had a greater incidence of non-Q-wave myocardial infarction (p less than 0.01) and ventricular premature contractions (p less than 0.01); they were more frequently in Moss class greater than 2 (p less than 0.01) and they had a lower wall motion score (p less than 0.05). At 2-year follow-up, patients with ST changes had a higher incidence of cardiac death and reinfarction. At multivariate analysis, Killip class (p less than 0.01) and ST changes (p less than 0.05) were the most predictive variables; when multivariate analysis was repeated including an additional variable--the inability to perform a stress test--Killip class was the most significant variable (p less than 0.01), and the presence of ST changes showed only borderline statistical significance (p less than 0.1). In the subset of patients who did not perform the stress test, ST change was the most important variable (p less than 0.01), followed by Killip class (p less than 0.05). Thus, after myocardial infarction, ST changes during Holter monitoring are associated with a poor prognosis and appear useful for stratifying patients who do not perform exercise stress tests.


Relationship between cell-cycle state of erythroid progenitors and fetal hemoglobin synthesis.

DNA-synthesis state of circulating burst forming unit-erythroid (BFU-E) was evaluated in patients with sickle cell anemia and correlated with percent of fetal hemoglobin synthesized in the BFU-E-derived cells. Percentage of S-phase BFU-E inversely correlated with percent fetal hemoglobin synthesized in the BFU-E-derived cells (simple linear correlation coefficient, r = -0.8, P = 0.0302; polynomial regression, R = -0.99, P = 0.0002). This observation is of relevance to our understanding of the relationship between the developmental stage of the erythroid progenitors and expression of globin genes.


Human monoclonal antibody recognizing an antigen associated with ovarian and other adenocarcinomas.

MS2B6, a human monoclonal antibody derived from a patient with advanced ovarian cancer, has been used to study the distribution and characteristics of its target antigen. The MS2B6 antigen was detected by immunoperoxidase studies in 41 of 41 epithelial ovarian cancers and in the majority of nonovarian adenocarcinomas. Among normal tissues the MS2B6 antigen was found in the adult epithelia of the fallopian tube, endometrium, endocervix, colon, bronchus, breast, sweat duct, and large renal ducts. No detectable antigen was found in peritoneal epithelia, tissue stromal cells, spleen, thymus, or blood-borne cells. Immunoblotting analysis showed that the MS2B6 epitope resides on polypeptides of 38, 44, and 60 kd. The cellular location of the MS2B6 antigen was studied with immunoperoxidase and immunofluorescent staining and immunoelectronmicroscopy of ovarian cancer ascites tumor cells. The results suggest that in ascites tumor cells the MS2B6 antigen is located in a layer of the peripheral cytoplasm beginning just below the cell membrane. MS2B6 may be useful as an imaging or therapeutic agent.


The carotenoid halocynthiaxanthin: a novel inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2.

We have studied the effects of a natural carotenoid, identified as halocynthiaxanthin, on the enzymatic activities associated with the recombinant preparations of the reverse transcriptases (RTs) of human immunodeficiency viruses (HIV) types 1 and 2. The carotenoid was found to be a potent inhibitor of the RNA-dependent DNA polymerase activity (with 50% inhibition obtained at 5-7 microM halocynthiaxanthin), whereas the DNA-dependent DNA polymerase function of both RTs was significantly less sensitive to the inhibitor. Conversely, the ribonuclease H activity associated with the two HIV RTs was essentially insensitive to the carotenoid. The RNA-dependent DNA polymerase function of RT is the only unique activity found in this enzyme that is not expressed at significant levels in uninfected eukaryotic cells. Therefore, it is possible that this carotenoid may serve as a good candidate for the development of novel potent and specific inhibitors of HIV RT.


Lymphoproliferative disorder of granular lymphocytes. A heterogeneous disease.

Lymphoproliferative disorders of granular lymphocytes (LDGLs) represent a family of diseases that are morphologically similar but diverse with regard to immunophenotype, function, and clonality. In this article, we report three informative cases and propose a modification of the current classification of LDGLs. Our first case is an example of natural killer cell LDGLs (CD2+, CD3-, CD16+, CD57+/-). Based on a review of the literature, we suggest that natural killer cell LDGLs can be divided into two subgroups (types 1 and 2) according to the expression of CD57. Reduced expression of CD57 may distinguish between patients with a poorer prognosis. The remaining two cases illustrate examples of T-cell LDGLs (CD2+, CD3+, CD8+, CD57+) that differ mainly in their expression of CD16. The CD16+ T-cell LDGLs (type 1) usually show a clonal rearrangement of the T-cell receptor-beta chain gene, whereas CD16- T-cell LDGLs (type 2) may show a germline configuration, suggesting a reactive rather than a neoplastic process. Pathologists should differentiate LDGLs from other chronic lymphoproliferative diseases, since most cases evolve slowly and aggressive cytoreductive therapy is usually unwarranted.