Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: heterogeneity and rate variations

Rates of fixation of mutations during the evolution of the foot-and-mouth disease virus (FMDV) C₁ in nature have been estimated by hybridization of viral RNA to cloned cDNAs representing defined FMDV genome segments, and comparison of the selected RNAs by T₁ RNase oligonucleotide fingerprinting. Values ranged from <0.04 × 10⁻² to 4.5 × 10⁻² substitutions per nucleotide per year (s/nt/yr), depending on the time period and the genomic segment considered. Rates for viral structural protein genes were up to sixfold higher than for nonstructural protein genes. Values in excess of 10⁻² s/nt/yr have been measured for the RNA region that encodes VP1–VP3. The nucleotide sequences of the major immunogenic region of capsid protein VP1 have been determined for six new FMDV C₁ isolates, and they are compared with the two previously known sequences of FMDV C₁ (C-S8 and C₁-O). Both oligonucleotide fingerprinting of selected RNA fragments and direct nucleotide sequencing demonstrate that genetic heterogeneity exists among three viruses isolated on the same day, introducing a significant indetermination in the evaluation of fixation rates of mutations. During the FMDV C₁ outbreak, amino acid substitutions did occur that are known to affect the immunological properties of the virus. The proportion of mutations between two viral RNAs does not increase significantly with the time elapsed between the two isolations, suggesting a cocirculation of multiple, related, nonidentical FMDVs (‘evolving quasispecies’) as the mode of evolution of this agent.     

Drosophila melanogaster contains a set of polypeptides capable of polymerizing into intermediate-like filaments

A partial purification scheme applied to the Triton X-100 insoluble pellet of adult flies homogenates yielded a fraction which upon polymerization reproducibly produces filaments which in the electron microscope have all the typical properties of intermediate filaments. This is the first report of the presence of protein components with such properties in Drosophila. Thus, it is highly possible that insects and arthropodes, like other lower invertebrates, may contain the third extremely insoluble element of the cellular cytoskeleton.     

Lymphatic drainage of Listeria monocytogenes and Indian ink inoculated in the peritoneal cavity of the mouse.

The lymphatic drainage of the peritoneal cavity has been investigated by intraperitoneal inoculation of an intracellular bacterium (Listeria monocytogenes) and an inert marker (Indian ink). The results reveal that both agents are transported, either after phagocytosis by intraperitoneal macrophages or in suspension in the lymph, towards the cranial sternal lymph nodes (Lymphonodi sternales craniales) of the ventral thoracic lymphocentrum (Lymphocentrum thoracicum ventrale) and to the lymph nodes of the mediastinal lymphocentrum (Lymphocentrum mediastinale), prior to systemic dissemination. This mechanism of peritoneal lymph drainage has relevance on experimental studies involving the inoculation of pathogens, and on the investigation of metastatic diffusion of neoplasms from the peritoneum.     

Host cell factors controlling vimentin organization in the Xenopus oocyte

To study vimentin filament organization in vivo we injected Xenopus oocytes, which have no significant vimentin system of their own, with in vitro-synthesized RNAs encoding Xenopus vimentins. Exogenous vimentins were localized primarily to the cytoplasmic surface of the nucleus and to the subplasma membrane "cortex." In the cortex of the animal hemisphere, wild-type vimentin forms punctate structures and short filaments. In contrast, long anastomosing vimentin filaments are formed in the vegetal hemisphere cortex. This asymmetry in the organization of exogenous vimentin is similar to that of the endogenous keratin system (Klymkowsky, M. W., L. A. Maynell, and A. G. Polson. 1987. Development (Camb.). 100:543-557), which suggests that the same cellular factors are responsible for both. Before germinal vesicle breakdown, in the initial stage of oocyte maturation, large vimentin and keratin filament bundles appear in the animal hemisphere. As maturation proceeds, keratin filaments fragment into soluble oligomers (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787-797), while vimentin filaments remain intact and vimentin is hyperphosphorylated. To examine the role of MPF kinase in the M-phase reorganization of vimentin we deleted the conserved proline of vimentin's single MPF-kinase site; this mutation had no apparent effect on the prophase or M-phase behavior of vimentin. In contrast, deletion of amino acids 19-68 or 18-61 of the NH2-terminal "head" domain produced proteins that formed extended filaments in the animal hemisphere of the prophase oocyte. We suggest that the animal hemisphere cortex of the prophase oocyte contains a factor that actively suppresses the formation of extended vimentin filaments through a direct interaction with vimentin's head domain. During maturation this "suppressor of extended filaments" appears to be inactivated, leading to the formation of an extended vimentin filament system.     

Immunohistological demonstration of pyruvate kinase isoenzyme type L in rat with monoclonal antibodies.

Four monoclonal antibodies (MAb) specific for the L-type isoenzyme of rat pyruvate kinase (L-PK) were produced and characterized. They detect at least two different epitopes of the isoenzyme, as shown in interference binding assay and Western blot analysis after peptide mapping. The MAb were used in immunohistology to demonstrate the L-PK isoenzyme in paraffin-embedded normal rat tissues. L-PK was found only in hepatocytes, kidney proximal tubules, islet cells of pancreas, and epithelial cells of the villi of small intestine. The content of L-PK in hepatocytes was often lower in the periportal areas as compared with the periveneous zone. In kidneys a clearcut difference in L-PK content and distribution existed between male and female rats. Male animals possessed more L-PK in the kidney cortex than females. The L-PK content in the inner cortical zone (straight proximal tubules) was higher than in the outer cortical zone (convoluted proximal tubules) in kidneys of males. In contrast, female rats displayed less L-PK in the inner than in the outer cortical zone of the kidneys. Only some of them exhibited the same amount of the isoenzyme in both parts of the kidney proximal tubules.     

Epitope mapping of the human biliary amphipathic, anionic polypeptide: similarity with a calcium-binding protein isolated from gallstones and bile, and immunologic cross-reactivity with apolipoprotein A-I.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of water stress and rewatering on leaf water relations of lemon plants

Potted two-year-old lemon plants (Citrus limon (L.) Burm. fil.) cv. Fino, growing under field conditions were subjected to drought by withholding irrigation for 13 d. After that, plants were re-irrigated and the recovery was studied for 5 d. Control plants were daily irrigated maintaining the soil matric potential at about -30 kPa. Young leaves of control plants presented higher leaf conductance (g1) and lower midday leaf water potential (Ψmd) than mature ones. Young leaves also showed higher leaf water potential at the turgor loss point (Ψtlp) than mature leaves. In both leaf types g1 decreased with increased vapour pressure deficit of the atmosphere. From day 1 of the withholding water, predawn and midday leaf water potentials (Ψpd and Ψmd) decreased, reaching in both cases minimum values of -5.5 MPa, with no significant differences between mature and young leaves. Water stress induced stomatal closure, leaf rolling and partial defoliation. No osmotic adjustment was found in response to water stress in either leaf type, but both were able to enhance the cell wall elasticity (elastic adjustment). After rewatering, leaf water potential recovered quickly (within 2 d) but g1 did not. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR

 DNA marker-assisted selection was used to pyramid four bacterial blight resistance genes, Xa-4, xa-5, xa-13 and Xa-21. Breeding lines with two, three and four resistance genes were developed and tested for resistance to the bacterial blight pathogen (Xanthomonas oryzae pv. oryzae). The pyramid lines showed a wider spectrum and a higher level of resistance than lines with only a single gene. To speed up the gene pyramiding process and to facilitate future marker-aided selection, we developed PCR markers for the two recessive genes, xa-5 and xa-13, and used these to survey a range of rice germplasm. The results of the germplasm survey will be useful for the selection of parents in breeding programs aimed at transferring these bacterial blight resistance genes from one varietal background to another. Received: 6 December 1996/Accepted: 20 December 1996     

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.     

Strong buffering capacity of insect cells. Implications for the baculovirus expression system

Insect cells are widely used for expression of a variety of different proteins by using the baculovirus expression system. The applicability of this system depends on production of proteins which have biological properties similar to their native counterparts. One application has been the expression of viral capsid proteins and their assembly into empty capsid structures to provide new viral immunogens which retain complex antigenic sites. An important parameter for efficient folding and assembly of proteins into viral procapsids may be the intracellular pH, particularly for acid-labile particles such as foot-and-mouth disease virus (FMDV). Benzoic acid was used as an effective indicator of intracellular pH in insect cells and 3-O-methyl glucose to measure cell volumes. We have determined the intracellular volume of theSpodoptera frugiperda IPLB-Sf21 insect cells 0.50±0.08 pL per cell. Using the distribution of [¹⁴C]-benzoic acid, we show that the intracellular pH remains constant at pH 7.0 when the cells are grown in media with pH values ranging from 6.2 to 6.8 and, moreover, is not affected by baculovirus infection. These results suggest that insect cells are suitable to express and produce acid-labile structures via the baculovirus expression system and that assembly of proteins and viral procapsids could occur.