Gas cascade amplification in ultra-high-resolution environmental scanning electron microcopy.

We describe a feedback mechanism in the gas cascade amplification process used in magnetic immersion lens environmental scanning electron microcopy (ESEM). Feedback dominates gas gain under the conditions typically used for ultra-high-resolution ESEM and gives rise to novel dependencies of the imaging signal and noise on microscope operating parameters. It is ascribed tentatively to the generation of free electrons upon de-excitation of metastable species in the gas cascade. The results have implications for optimization of ESEM systems for applications such as critical dimension metrology and real-time imaging of nanostructure growth by gas mediated electron beam induced deposition.

Spatiotemporal segregation of endothelial cell integrin and nonintegrin extracellular matrix-binding proteins during adhesion events.

Bovine aortic endothelial cell (BAEC) attachments to laminin, fibronectin, and fibrinogen are inhibited by soluble arginine-glycine-aspartate (RGD)-containing peptides, and YGRGDSP activity is responsive to titration of either soluble peptide or matrix protein. To assess the presence of RGD-dependent receptors, immunoprecipitation and immunoblotting studies were conducted and demonstrated integrin beta 1, beta 3, and associated alpha subunits as well as a beta 1 precursor. Immunofluorescence of BAECs plated on laminin, fibronectin, and fibrinogen reveals different matrix-binding specificities of each of these integrin subclasses. By 1 h after plating, organization of beta 1 integrin into fibrillar streaks is influenced by laminin and fibronectin, whereas beta 3 integrin punctate organization is influenced by fibrinogen and the integrin spatial distribution changes with time in culture. In contrast, the nonintegrin laminin-binding protein LB69 only organizes after cell-substrate contact is well established several hours after plating. Migration of BAECs is also mediated by both integrin and nonintegrin matrix-binding proteins. Specifically, BAEC migration on laminin is remarkably sensitive to RGD peptide inhibition, and, in its presence, beta 1 integrin organization dissipates and reorganizes into perinuclear vesicles. However, RGD peptides do not alter LB69 linear organization during migration. Similarly, agents that block LB69--e.g., antibodies to LB69 as well as YIGSR-NH2 peptide--do not inhibit attachment of nonmotile BAECs to laminin. However, both anti-LB69 and YIGSR-NH2 inhibit late adhesive events such as spreading. Accordingly, we propose that integrin and nonintegrin extracellular matrix-binding protein organizations in BAECs are both temporally and spatially segregated during attachment processes. High affinity nonintegrin interaction with matrix may create necessary stable contacts for longterm attachment, while lower affinity integrins may be important for initial cell adhesion as well as for transient contacts of motile BAECs.

Intracellular study of human epileptic cortex: in vitro maintenance of epileptiform activity?

Intracellular recordings were obtained in the vitro slice preparation from neurons of lateral and mesial temporal cortex removed from human epileptics suffering from intractable temporal lobe seizures. Spontaneous rhythmic synaptic events, which were capable of triggering action potential discharge, were observed in many neurons, particularly in mesial tissue slices. Such activity may reflect the epileptogenic capacity of this human cortex.

Common structural polymorphisms in human erythrocyte spectrin.

Restricted tryptic digestion of erythrocyte spectrin at 4 degrees C followed by two-dimensional (isoelectric-focusing/sodium dodecyl sulfate) polyacrylamide electrophoresis yields highly reproducible maps of approximately 50 peptides with molecular weights between 80,000 and 12,000. Based on molecular weight and isoelectric point (pI), each unique alpha- and beta-subunit domain can be identified and compared with spectrin peptides from other individuals. The alpha-subunit of spectrin from 60 Caucasian donors contains a 46,000-mol-wt tryptic domain, called alpha II-T46, Type 1; more extensive tryptic digestion of this domain generates peptides with molecular weights of 35,000, 30,000, 25,000, and 16,000. Spectrin from 29 of 37 black donors representing 14 kindreds shows variation in the molecular weight and/or pI of peptides from the alpha II domain. In the most common form, Type 2, alpha II tryptic peptides are increased in molecular weight by 4,000, and the pI becomes more basic. Other alpha II variants are characterized by either the 4,000 increase in molecular weight (Type 3) or by the basic shift in pI (Type 4). When limit peptide maps of intermediate-sized tryptic and CNBr peptides from the alpha II-domain Types 1 and 2 are compared, a consistent alteration in the chromatographic mobility of one limit peptide is observed. Polymorphism in the alpha II subunit of spectrin did not itself produce anemia, nor did it appear to alter the expression of an underlying hereditary spherocytosis or elliptocytosis. In six family studies, the alpha II 46,000-mol-wt variations observed were consistent with Mendelian inheritance.

Molecular and functional changes in spectrin from patients with hereditary pyropoikilocytosis.

The structural and functional properties of spectrin from normal and hereditary pyropoikilocytosis (HPP) donors from the two unrelated families were studied. The structural domains of the spectrin molecule were generated by mild tryptic digestion and analyzed by two-dimensional electrophoresis (isoelectric focusing; sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The alpha I-T80 peptide (Mr 80,000) is not detectable in two related HPP donors; instead, two new peptides (Mr 50,000 and 21,000) are generated and have been identified as fragments of the normal alpha I-T80. A third sibling has reduced levels of both the normal alpha I-T80 and the two new peptides. A similar analysis of spectrin from another HPP family indicates that their spectrins contain reduced amounts of the alpha I-T80 and the 50,000 and 21,000 fragments of the alpha I domain. The HPP donor also has other structural variations in the alpha I, alpha II, and alpha III domains. The alpha I-T80 domain of normal spectrin has been shown to be an important site for spectrin oligomerization (J. Morrow and V.T. Marchesi. 1981. J. Cell Biol. 88: 463-468), and in vitro assays indicate that HPP spectrin has an impaired ability to oligomerize. Ghost membranes from HPP donors are also more fragile than membranes from normal erythrocytes when measured by ektacytometry. In both the oligomerization and fragility assays, the degree of impairment is correlated with the amount of normal alpha I-T80 present in the spectrin molecule. We believe that a structural alteration in the alpha I-T80 domain perturbs normal in vivo oligomerization of spectrin, producing a marked decrease in erythrocyte stability.

Antibodies to glutamic acid decarboxylase discriminate major types of diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is marked by circulating antibodies to a 64,000-M(r) islet cell antigen identified as glutamic acid decarboxylase (GAD). We describe a radioimmunoprecipitation assay with GAD isolated from pig brain. The sera tested were from 80 patients with IDDM including 26 with disease of recent onset and 54 with disease of longer duration (3-42 yr), 20 with non-insulin-dependent diabetes mellitus (NIDDM), and 55 nondiabetic subjects. Conventional assays for islet cell cytoplasmic antibodies were performed concurrently. The level of antibody in serum was expressed in units based on percentage reactivity of a standard reference serum. The frequency of antibody to GAD in IDDM was 69% in short-duration cases and 59% in long-duration cases. The latter was substantially higher than the frequency of islet cell cytoplasmic antibody. Antibodies to GAD were elevated (means +/- 3 SD) in 5% NIDDM cases and in none of the nondiabetic subjects. A simple laboratory test with a defined autoantigen has substantial implications for population screening and early diagnosis of IDDM and for better understanding of its pathogenesis.

Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes mellitus in adults with a non-insulin-dependent onset of disease.

The classification of adults with diabetes mellitus can be invalidated by patients who initially present as NIDDM but who later become frankly insulin dependent. In some of these, the pathogenesis could be similar to that in IDDM, namely autoimmune destruction of the pancreatic beta-cells. We studied 102 patients > 35 yr of age at diabetes onset who had initially been nonketotic and non-insulin-dependent for > or = 6 mo. They were classified according to glucagon-stimulated C-peptide levels into an insulin-deficient group (n = 33) and a non-insulin-deficient group (n = 69). We measured antibodies to GAD, islet cell cytoplasm, thyroid antigens, and gastric parietal cells in both groups. Anti-GAD was significantly higher in the insulin deficient group, 76% (25 of 33), than in the non-insulin deficient group, 12% (8 of 69), and this difference was substantially greater than that shown for ICAs. Thus, in a proportion of adults who present with NIDDM, a slowly evolving autoimmune insulitis can be revealed by testing for anti-GAD. This could have important connotations not only for early intervention, but also for the correct classification of diabetes.

Neuronal responses to angiotensin II in the in vitro slice from the canine medulla.

The present studies utilized the in vitro slice preparation of the canine dorsomedial medulla, which we have recently developed, to obtain direct evidence for the effects of angiotensin II (Ang II) on the activity of single neurons in this region. Horizontally oriented slices (300 micron) containing the area postrema, nucleus tractus solitarii (NTS), and dorsal motor nucleus of the vagus were perifused with oxygenated artificial cerebrospinal fluid. The effects of microdrop application of Ang II and its antagonist [Sar1,Thr8]Ang II on spontaneous firing rate were determined in 27 extracellularly recorded neurons. Ang II substantially increased the firing rate of 13 neurons located in the medial NTS, but it did not alter the spontaneous activity of the remaining 14 neurons. In most cases Ang II elicited a slowly developing, prolonged excitatory response. The effects of both Ang II and [Sar1,Thr8]Ang II were tested in 13 neurons. [Sar1,Thr8]Ang II produced a short latency, brief excitation in three neurons, marked inhibition of spontaneous firing in two cells, and no effect on the other eight neurons. Administration of [Sar1,Thr8]Ang II blocked the excitatory effects of subsequent administration of Ang II in three neurons. To our knowledge, these observations provide the first evidence for direct actions of both Ang II and [Sar1,Thr8]Ang II on neurons in the canine NTS and for the specificity of the neuronal effects of Ang II as documented by blockade of the excitatory response to Ang II by [Sar1,Thr8]Ang II.

Spectrin: structure, function, and abnormalities.

A number of proteins that make up the membrane skeleton have been identified, and we have a rough but tantalizing picture of the ways in which they interact to maintain its integrity. An approximate molecular model of spectrin has been proposed, and many new analytic procedures have been developed to search for biochemical variants that may be related to membrane defects. Even at this rudimentary stage of molecular description we have been able to identify structural changes in spectrin that are correlated with pathophysiologic states. The prospects for some genuine insights into the pathogenesis of some congenital hemolytic anemias seem good.

The initiation and spread of epileptiform bursts in the in vitro hippocampal slice.

We recorded spontaneous synchronized epileptiform bursts from hippocampal slices from guinea pig using an array of 16 extracellular electrodes placed over the stratum pyramidale of CA2 and CA3. The slices were made epileptogenic with the GABA antagonist picrotoxin (or occasionally penicillin). We found that spontaneous bursts always originate at a discrete focus at or near CA2. These bursts spread smoothly and uniformly across CA3 at an average velocity of 0.13 m/s. This velocity is slower than the conduction velocity of the Schaffer collaterals or mossy fibers. Picrotoxin produced afterdischarges following the initial primary burst, and these afterdischarges were found to originate and spread in a fashion nearly identical to the primary burst. These results indicate that CA2 is a unique region which must possess unusual cellular and/or synaptic connectivity properties which result in a decreased threshold for initiation of epileptiform activity. We consider several hypothetical patterns of local synaptic connectivity in the light of these results, and we discuss the possible role of residual inhibition in limiting the spread of synchronized discharges.

Models of the cellular mechanism underlying propagation of epileptiform activity in the CA2-CA3 region of the hippocampal slice.

We have shown experimentally in the previous paper that spontaneous epileptiform activity, as recorded by extracellular field potentials, propagates smoothly across the CA2-CA3 region of the convulsant-treated hippocampal slice of the guinea pig at velocities of about 0.1 m/s. In the present paper, we used computer simulations of either 500 or 1000 cell arrays of model neurons to examine possible mechanisms underlying this propagation. We show that propagation of epileptiform field potentials can be explained plausibly by slow conduction along axons interconnecting CA2-CA3 neurons, provided that there are sufficiently many interconnections. This propagation can take place even if the interconnections occur randomly. The number of interconnections required decreases as the number of synchronously activated cells initiating a population burst increases. Axonal propagation at 0.1 m/s appears to be a plausible assumption, since conduction velocities along Schaffer collaterals have been experimentally estimated to be as slow as 0.2 m/s, and small recurrent collaterals are likely to conduct more slowly than the main axonal branches. If spontaneous synchronized population bursts are initiated by activity in four or fewer cells, then our model requires, for smooth field potential propagation, more interconnections than are believed to occur on the basis of dual intracellular recording.

Synchronized afterdischarges in the hippocampus: simulation studies of the cellular mechanism.

Synchronized multiple bursts represent an epileptic neuronal behavior transitional between synchronized single bursts (interictal spikes) and self-sustained seizures. As described in the previous paper, synchronized multiple bursts occur in hippocampal slices treated with picrotoxin. Multiple bursts consist of an initial prolonged depolarizing burst followed by a rhythmical series of afterdischarges. Both the initial burst and the afterdischarges are synaptically elicited. Our previously described model of the interictal spike illustrates that the generation of a single synchronized burst requires a neuronal network possessing the following properties: intrinsic bursting capability of individual neurons, the presence of recurrent excitatory connections between principal neurons and the blockade of synaptic inhibition. The model demonstrates that the generation of single synchronized bursts involves the initial excitation of one or more neurons, and the subsequent sequential spread of excitation through a population of neurons via recurrent excitatory synapses. In the present study, we examined whether this same mechanism assumed in the previous model could also allow for the generation of synchronized afterdischarges in a population of neurons. We tested the effects of manipulating three network factors: synaptic strength, synaptic density and the refractoriness in the population members following a period of excitation. We discovered that the refractory period following prolonged excitation assumed in our previous model was insufficient to allow for afterdischarge generation. Once sufficient refractoriness was introduced, afterdischarges appeared in our network of neurons. In the present study, the required refractoriness was attributed to the properties of pyramidal cell axons. In principle, such refractoriness might be located elsewhere in the network. The possible contribution of axonal properties is emphasized because of the known intermittent conduction in other axons. Our present model also reproduced other experimental data. Thus, if the network was too small or if synaptic strength was too small, then only a single synchronized burst occurred. The basic assumptions of this model are both biologically plausible and experimentally testable.

Electrotonic and dye coupling in hippocampal CA1 pyramidal cells in vitro.

The presence of electrotonic and dye coupling in region CA1 of the guinea-pig hippocampus was investigated in the in vitro hippocampal slice preparation. No electronic coupling potentials were observed in simultaneous recordings from 101 pairs of pyramidal cells. Also, no electrotonically-coupled short latency depolarizations were observed in more than 75 pyramidal cells in response to antidromic activation of the pyramidal cell population, either in normal bathing medium or in medium with lowered Ca2+ concentration and added Mn2+. When the fluorescent dye Lucifer Yellow was injected into pyramidal cell somas, spread of the dye to other cells (dye coupling) was often observed. Injection of Lucifer Yellow into the dendrites of these neurons resulted in many fewer cases of dye coupling. The failure to find electrophysiological evidence of electrotonic coupling among CA1 pyramidal cells suggests that such coupling is not a functionally important feature of this area of the CNS. The lack of electrophysiological evidence of coupling combined with the observation that the site of Lucifer Yellow injection influences the extent of dye coupling further suggests that at least part of the observed dye coupling may be artifactual. Electrotonic coupling may exist in a small percentage of hippocampal pyramidal cells. However, it is not clear that this small amount of coupling is either necessary or sufficient for the synchronization of neural activity as has been hypothesized to occur during epileptogenesis.

Computer simulations indicate that electrical field effects contribute to the shape of the epileptiform field potential.

In the presence of convulsant drugs such as picrotoxin, neurons in the hippocampal-slice preparation generate synchronized depolarizing bursts. This synchrony occurs on a time scale of tens of milliseconds and is produced by excitatory synaptic interactions between neurons. The synaptic interactions themselves occur on a time scale of tens of milliseconds. The "epileptiform" local-field potential during such synchronized bursts is comb-shaped ("ringing"), whereas the field potential expected if action potentials in neighboring neurons were uncorrelated is noisy and not comb-shaped. This suggests that individual action potentials are locally synchronized on a time scale of 1 ms. We have previously shown, using computer simulations, that electrical interactions--mediated by currents flowing in the extracellular medium--can plausibly explain action-potential synchronization in experiments where chemical synapses are blocked. The present simulations demonstrate that electrical interactions can also account for action-potential synchronization--and thus the "ringing" shape of the field potential--during epileptiform bursts, where excitatory synapses are functional. The field potential is thus a modulating influence on, as well as a reflection of, underlying neuronal transmembrane events.

Simulation of hippocampal afterdischarges synchronized by electrical interactions.

Recent experiments have shown that hippocampal pyramidal cells can generate synchronized action potentials even when chemical synapses are blocked. The computer simulations reported here showed that communication between cells by extracellular currents could cause this synchrony, provided that (1) individual neurons were sufficiently excitable and that (2) the resistivity of the extracellular medium was sufficiently high. Synchronization was enhanced if electronic junctions were also present.

The genomic sequence of a contemporary wild-type mumps virus strain.

Vaccination has the potential to eradicate mumps, and 82 countries now include a live attenuated mumps vaccine as part of their childhood vaccination programme. Although, monotypic, genetic variants of mumps virus (MuV) have been described based on comparison of the SH gene sequences, and at least seven genotypes have been identified. We now report the entire sequence of a recently isolated wild type MuV strain, Glouc1/UK96 (Glouc1) by direct sequencing of the cDNA obtained from cell culture fluid. The genome of this recent isolate was 15384 nucleotides in length. There were 579 nucleotide differences (3.8%) and 71 amino acid differences (1.5%) between Glouc1, a genotype G strain and Ur-AM9, a genotype B strain. Other MuV strains with available sequences were also compared with this pathological strain. The sequence of the contemporary strain reported here provides a picture of the variability of MuV over its entire genome (GenBank accession no. AF280799).

Genetic and antigenic characterisation of the haemagglutinin protein of measles virus strains recently circulating in the UK.

The complete nucleotide sequence of the H protein gene of seven measles virus (MV) strains, representing three MV genotypes circulating in the UK in recent years, was determined. Compared to the MV vaccine strain Moraten (Mor-v), the divergence of the coded H gene (aal-600) of the seven UK strains was between 1.8% and 2.8%. Representative isolates from each of the genotypes were tested by radio-immunoprecipitation using a panel of H protein-specific MAbs. Different patterns of MAb reactivity were shown between the three genotypes and between the wild-type strains and the vaccine strain. Plaque reduction neutralising antibody titres against strains UK350/94 (genotype I) and UK226/94 (genotype III) were measured in sera from 11 vaccinees. Vaccine derived antibody neutralised both strains and the GMTs were not significantly lower against the wild-type strains than against strain Mor-v.

Successful treatment of parvovirus B19 infection and red cell aplasia occurring after an allogeneic bone marrow transplant.

Chronic parvovirus B19 infection in the immunocompromised host may cause severe anaemia secondary to failure of erythropoiesis. This has been previously documented in patients with the Acquired Immune Deficiency Syndrome (AIDS), congenital immunodeficiencies and in children with acute lymphoblastic leukaemia during maintenance chemotherapy. We describe persistent parvovirus infection in a 14-year-old boy after HLA-matched sibling allogeneic bone marrow transplantation for acute lymphoblastic leukaemia in second remission.

The identification of GABA receptor binding sites in insect ganglia.

The binding of [(3)H]muscimol to membrane fractions from central nervous tissue from fly (Musca domestica) and locust (Schistocerca gregaria) was measured. Specific saturable binding was observed with K(d)s of 10 nM (locust) and 40 nM (fly) and B(max) values of 20 fmol/mg protein (fly) and 60 fmol/mg protein (locust). Binding was Na(+)-independent and in locust tissue an additional Na(+)-dependent binding component was observed. Pharmacological properties of the binding sites in both species are consistent with a GABA receptor site rather than an uptake/transport site. We conclude that insect central nervous tissue contains receptors that show some similarities to the GABA receptor complex of mammalian brain.

Viral exposure and abnormal liver function in haemophilia.

Several studies have recently documented the presence of persistently abnormal liver function tests in asymptomatic haemophiliacs. While the aetiology is unknown it is possible that repeated exposure to agents transmitted in blood products may be important. This study has attempted to determine the prevalence of viral exposure and its relationship to liver function in this multitransfused group of individuals. The prevalence of viral antibodies with the exception of antibody to hepatitis B surface antigen (anti-HBs) and cytomegalovirus (CMV) was normal when compared to that in the general population. Hepatitis B surface antigen (HBsAg) was not detected, but anti-HBs was found in 83% of patients; 50% of patients had abnormal liver function. However, liver function tests were normal in all patients with mild haemophilia and were only rarely abnormal in patients who had no detectable antibody to CMV, Epstein-Barr virus (EBV), and HBsAg. This study demonstrates that multiple transfusions of blood products, that is, cryoprecipitate and factor concentrates, do not increase the risk of exposure to the viruses studied with the exception of hepatitis B virus.