A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.     

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney

High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg²⁺, Mn²⁺, and Co²⁺ each supported activity, but highest activity was obtained with Mg². The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E_a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μm. The K_m for cyclic AMP hydrolysis was 2 μm, and that for cyclic GMP hydrolysis was 312 μm. The K_i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K_m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K_i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.     

Regeneration of changes produced in rabbit tracheal epithelium by saline lavage of the airways

The authors studied the course of the repair of changes induced in the rabbit tracheal epithelium by saline lavage of the airways. The tracheal epithelium was examined 2, 24, 48 and 72 hours after treatment. Saline lavage stimulated the goblet cells to instantaneous discharge of their secretion. 2 hours after treatment 98 +/- 3% of the goblet cells were completely exhausted and had degenerated. Repair of the changes began 24 hours after lavage and was associated with massive differentiation of new goblet cells resulting in hyperplasia of the mucus-secreting elements with formation of endoepithelial mucous glands. The most pronounced injury to the ciliated cells was apparent 2 hours after lavage, then the degree of alteration of these cells gradually decreased. Saline lavage markedly impaired the ciliary border. The mean number of kinocilia per micron2 fell to 1.5 +/- 0.3. In subsequent phases the number of kinocilia rose gradually to 7.5 +/- 0.5/micron2. This value was still significantly lower (P less than 0.005) compared with controls. The first signs of impairment of the self-cleaning ability of the epithelium were recorded 2 hours after lavage. The most pronounced disturbances of the mucus flow were observed after 24 hours. At the end of the experimental period small clumps of condensed mucus and rather numerous bacteria were still present in the area of the ciliary border.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.     

The study of genetic diversity patterns of Coffea commersoniana, an endangered coffee species from Madagascar: a model for conservation of other littoral forest species

Madagascar has 59 described species of Coffea, of which 42 are listed as critically endangered, endangered, or vulnerable by the criteria of the Red List Category system of the World Conservation Union (IUCN). The littoral forest of Madagascar is a distinctive type of humid evergreen forest restricted to unconsolidated sand located within a few kilometers of the Indian Ocean, now persisting only as small fragments with ca. 10 % of its original range remaining. In an attempt to understand the genetic diversity of Madagascan coffee species, we studied ex situ and in situ populations of Coffea commersoniana, an endemic species of the littoral forests of southeastern Madagascar and soon to be impacted by mining activities in that region. The in situ populations studied showed higher genetic diversity than the ex situ population. The genetic partitioning among the two in situ populations of C. commersoniana was high enough to necessitate keeping the two populations separate for restoration purposes. Based on these findings, recommendations for conservation management (in situ and ex situ) are made.     

Major vault protein (MVP) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis

The human major vault protein (MVP) has been implicated in the development of drug resistance in cancer cells. Over expression of MVP has also been reported in brain tissue samples from antiepileptic drug (AED)-resistant human focal epilepsies. To investigate the relationship between single nucleotide polymorphisms (SNPs) involving the MVP gene and AED-resistance, we compared the distribution of three SNPs in the MVP gene, rs4788187, rs3815824 and rs3815823, among 220 patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype of AED-resistant epilepsy syndrome), 201 patients with juvenile myoclonic epilepsy (JME) (prototype of AED-responsive epilepsy syndrome) and 213 ethnically matched non-epilepsy controls. All the patients and controls were residents of the South Indian state of Kerala for more than three generations. We did not find any significant difference in allele and genotypic frequencies of the studied SNPs between AED-resistant and AED-responsive cohorts, and between AED-resistant and AED-responsive cohorts independently and pooled together when compared with the controls. We conclude that rs4788187, rs3815824, rs3815823 variants of the MVP gene are associated neither with predisposition for epilepsy nor with AED-resistance in the population that we have studied. Our results suggest the need for further research into the link between MVP and AED-resistance.     

Patient oriented graph-based image segmentation

Despite increased image quality including medical imaging, image segmentation continues to represent a major bottleneck in practical applications due to noise and lack of contrast. In this paper, we present a new methodology to segment noisy, low contrast medical images, with a view to developing practical applications. Firstly, the contrast of the image is enhanced and then a modified graph-based method is followed. This paper has mainly two contributions: (1) a contrast enhancement stage performed by suitably utilizing the noise present in the medical data. This step is achieved through stochastic resonance theory applied in the wavelet domain and (2) a new weighting function is proposed for traditional graph-based approaches. Both qualitative (by our clinicians/radiologists) and quantitative evaluation performed on publicly available computed tomography (CT) (MICCAI 2007 Grand Challenge workshop database) and cardiac magnetic resonance (CMR) databases reflect the potential of the proposed method even in the presence of tumors/papillary muscles.     

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.