Tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle.

Isolates of foot-and-mouth disease virus (FMDV) exist as complex mixtures of variants. Two different serotype O1 Campos preparations that we examined contained two variants with distinct plaque morphologies on BHK cells: a small, clear-plaque virus that replicates in BHK and CHO cells, and a large, turbid-plaque virus that only grows in BHK cells. cDNAs encoding the capsids of these two variants were inserted into a genome-length FMDV type A12 infectious cDNA and used to produce chimeric viruses that exhibited the phenotype of the original variants. Analyses of these viruses, and hybrids created by exchanging portions of the capsid gene, identified codon 56 in VP3 (3056) as the critical determinant of both cell tropism and plaque phenotype. Specifically, the CHO growth/clear-plaque phenotype is dependent on the presence of the highly charged Arg residue at 3056, and viruses with this phenotype and genotype were selected during propagation in tissue culture. The genetically engineered Arg 3056 virus was highly attenuated in bovines, but viruses recovered from animals inoculated with high doses of this virus had lost the ability to grow in CHO cells and contained either an uncharged residue at 3056 or a negatively charged Glu substituted for a Lys at a spatially and antigenically related position on VP2 (2134). Comparison of these animal-derived viruses to other natural and engineered viruses demonstrated that positively charged residues are required at both 2134 and 3056 for binding to heparin. Taken together, these results indicate that in vitro cultivation of FMDV type O selects viruses that bind to heparin and that viruses with the heparin-binding phenotype are attenuated in the natural host.     

Analysis of foot-and-mouth disease virus-neutralizing idiotypes from immune bovine and swine with anti-murine idiotype antibody probes

Rabbit anti-idiotypic antibodies (a-IdAb) induced by foot-and-mouth disease virus (FMDV) neutralizing mAb were used as probes to identify anti-FMDV Id in immune serum from bovine and swine. In a competitive RIA, at least two of the a-IdAb exhibited a dose-dependent capacity to compete with labeled virus for anti-FMDV antibodies from a convalescent bovine serum. These a-IdAb were immobilized on activated Sepharose and used to isolate anti-viral Id from bovine, swine, and murine FMDV immune sera. Both the bovine and swine antibodies recovered from the a-IdAb/Sepharose columns reacted with virus, and to a lesser extent with corresponding mAb-resistant virus variants. The binding of affinity isolated bovine and swine antibodies to virus was specifically inhibited by the homologous a-IdAb, and in addition, both were capable of neutralizing FMDV in suckling mouse protection and plaque reduction neutralization assays. Therefore, by means of a-IdAb probes generated against FMDV murine Id, two neutralizing Id were identified in bovine and swine. These results suggest that FMDV-neutralizing epitopes recognized by murine systems play a role in the overall immunity of foot-and-mouth disease-susceptible animals.     

OSMOPHORES OF STANHOPEA (ORCHIDACEAE)

Species of the Neotropical orchid genus Stanhopea produce a fragrance comprising terpenoids and aromatics which attracts euglossine bee pollinators. The secretory tissue, called an osmophore, is located in the adaxial region of a sac formed near the proximal portion of the floral lip. This region is easily recognized in Stanhopea oculata and S. wardii because it is papillate. The osmophore in these two species includes all the cells of the papillae and those directly below, that grade into fundamental tissue. Osmophore cells are more densely cytoplasmic than cells in the adjacent tissue. Numerous amyloplasts and mitochondria are seen in these cells from the earliest bud stages we examined through anthesis. Smooth and rough endoplasmic reticulum are abundant, but dictyosomes are uncommon. Mitochondria of osmophore cells appear to be distributed with no apparent pattern during bud stages, although they tend to be aligned near the plasmalemma at anthesis. Osmophore cells are highly vacuolate after anthesis.     

Isolation and Characterization of Mouse Minisatellites

Minisatellites provide the most informative system for analyzing processes of tandem repeat turnover in humans. However, little is known about minisatellites and the mechanisms by which they mutate in other species. To this end, we have isolated and characterized 76 endogenous mouse VNTRs. Fifty-one loci have been localized on mouse chromosomes and, unlike in humans, show no clustering in proterminal regions. Sequence analysis of 25 loci revealed the majority to be authentic minisatellites with GC-rich repeat units ranging from 14 to 47 bp in length. We have further characterized 3 of the most polymorphic loci both inMus musculussubspecies and in inbred strains by using minisatellite variant repeat mapping (MVR) by PCR to gain insight into allelic diversity and turnover processes. MVR data suggest that mouse minisatellites mutate mainly by intra-allelic nonpolar events at a rate well below 10⁻³per gamete, in contrast to the high-frequency complex meiotic gene conversion-like events seen in humans. These results may indicate a fundamental difference in mechanisms of minisatellite mutation and genome turnover between mice and humans.     

Hemodynamic correlates of effective arterial elastance in mitral stenosis before and after balloon valvotomy

Colin, Patrice, Michel Slama, Alec Vahanian, YvesLecarpentier, Gilbert Motté, and Denis Chemla. Hemodynamiccorrelates of effective arterial elastance in mitral stenosis beforeand after balloon valvotomy. J. Appl.Physiol. 83(4): 1083-1089, 1997.This study hadthe purpose of documenting the hemodynamic correlates of effectivearterial elastance (Ea; i.e., an accurate estimate of hydraulic load)in mitral stenosis (MS) patients. The main hypothesis tested was thatEa relates to the total vascular resistance (R)-to-pulse intervalduration (T) ratio(R/T) in MS patients both before andafter successful balloon mitral valvotomy (BMV). High-fidelity aorticpressure recordings were obtained in 10 patients (40 ± 12 yr)before and 15 min after BMV. Ea value was calculated as the ratio ofthe steady-state end-systolic aortic pressure (ESAP) to stroke volume(thermodilution). Ea increased after BMV (from 1.55 ± 0.63 to 1.83 ± 0.71 mmHg/ml; P < 0.05). Throughout the procedure, there was a strong linearrelationship between Ea and R/T: Ea = 1.09R/T  0.01 mmHg/ml,r = 0.99, P = 0.0001. This ultimately dependedon the powerful link between ESAP and mean aortic pressure [MAP;r = 0.99, 95% confidence interval for the difference (MAP  ESAP) from 18.5 to +4.5 mmHg].Ea was also related to total arterial compliance (area method) and towave reflections (augmentation index), although to a lesser extent. After BMV, enhanced and anticipated wave reflections were observed, andthis was likely to be explained by decreased arterial compliance. Thepresent study indicated that Ea depended mainly on the steady componentof hydraulic load (i.e., R) and on heart period (i.e., T) in MS patients.

     

Heparan sulfate-binding foot-and-mouth disease virus enters cells via caveola-mediated endocytosis

Foot-and-mouth disease virus (FMDV) utilizes different cell surface macromolecules to facilitate infection of cultured cells. Virus, which is virulent for susceptible animals, infects cells via four members of the alpha(V) subclass of cellular integrins. In contrast, tissue culture adaptation of some FMDV serotypes results in the loss of viral virulence in the animal, accompanied by the loss of virus' ability to use integrins as receptors. These avirulent viral variants acquire positively charged amino acids on surface-exposed structural proteins, resulting in the utilization of cell surface heparan sulfate (HS) molecules as receptors. We have recently shown that FMDV serotypes utilizing integrin receptors enter cells via a clathrin-mediated mechanism into early endosomes. Acidification within the endosome results in a breakdown of the viral capsid, releasing the RNA, which enters the cytoplasm by a still undefined mechanism. Since there is evidence that HS internalizes bound ligands via a caveola-mediated mechanism, it was of interest to analyze the entry of FMDV by cell-surface HS. Using a genetically engineered variant of type O(1)Campos (O(1)C3056R) which can utilize both integrins and HS as receptors and a second variant (O(1)C3056R-KGE) which can utilize only HS as a receptor, we followed viral entry using confocal microscopy. After virus bound to cells at 4 degrees C, followed by a temperature shift to 37 degrees C, type O(1)C3056R-KGE colocalized with caveolin-1, while O(1)C3056R colocalized with both clathrin and caveolin-1. Compounds which either disrupt or inhibit the formation of lipid rafts inhibited the replication of O(1)C3056R-KGE. Furthermore, a caveolin-1 knockdown by RNA interference also considerably reduced the efficiency of O(1)C3056R-KGE infection. These results indicate that HS-binding FMDV enters the cells via the caveola-mediated endocytosis pathway and that caveolae can associate and traffic with endosomes. In addition, these results further suggest that the route of FMDV entry into cells is a function solely of the viral receptor.     

Sympathetic input modulates, but does not determine, phase of peripheral circadian oscillators

The circadian clock in the suprachiasmatic nucleus (SCN) maintains phase synchrony among circadian oscillators throughout the organism. Environmental light signals entrain the SCN, but timed, limited meal access acts as an overriding time cue for several peripheral tissues. We present data from a peripheral oscillator, the submaxillary salivary gland, in which temporal restriction of meals fails to entrain gene expression. In day-fed rats, submaxillary gland rhythms in expression of the clock gene Period1 (Per1) stay entrained to the light cycle (peaking at night) or become arrhythmic. This result suggests that feeding cues compete weakly with light cycle cues to set the phase of clock genes in this tissue. Since the submaxillary glands receive sympathetic innervation originating in the SCN, which relays light cycle cues to other oscillators, we attempted to assess the role of this neural input in phase control of submaxillary Per1 expression. We sympathetically denervated the submaxillary glands before subjecting rats to daytime-restricted feeding. After denervation, Per1 rhythms in all submaxillary glands shifted phase 180 degrees and entrained to daytime feeding. These results support the hypothesis that peripheral oscillators may receive multiple signals contributing to their phase of entrainment. Sympathetic efferents from the SCN can relay light cycle information, while other external cues may reach tissues through other efferents or nonneural pathways. In an abnormal, disruptive regimen such as daytime-restricted feeding, these different signals compete. Arrhythmicity may result if one signal is not clearly dominant. Elimination of the dominant signal (e.g., surgical sympathectomy) may allow a secondary signal to control phase.