Hispin,a novel ribosome inactivating protein with antifungal activity from hairy melon seeds

A ribosome inactivating protein demonstrating a molecular mass of 21 kDa and a novel N-terminal sequence was isolated from seeds of the hairy melon. The purification procedure involved affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose and Mono S. The protein designated hispin inhibited translation in the cell-free rabbit reticulocyte lysate system with an IC50 of 165 pM and exhibited N-glycosidase activity. Antifungal activity was also observed.     

A novel alkaline alpha-galactosidase from melon fruit with a substrate preference for raffinose

The cucurbits translocate the galactosyl-sucrose oligosaccharides raffinose and stachyose, therefore, alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) is expected to function as the initial enzyme of photoassimilate catabolism. However, the previously described alkaline alpha-galactosidase is specific for the tetrasaccharide stachyose, leaving raffinose catabolism in these tissues as an enigma. In this paper we report the partial purification and characterization of three alpha-galactosidases, including a novel alkaline alpha-galactosidase (form I) from melon (Cucumis melo) fruit tissue. The form I enzyme showed preferred activity with raffinose and significant activity with stachyose. Other unique characteristics of this enzyme, such as weak product inhibition by galactose (in contrast to the other alpha-galactosidases, which show stronger product inhibition), also impart physiological significance. Using raffinose and stachyose as substrates in the assays, the activities of the three alpha-galactosidases (alkaline form I, alkaline form II, and the acid form) were measured at different stages of fruit development. The form I enzyme activity increased during the early stages of ovary development and fruit set, in contrast to the other alpha-galactosidase enzymes, both of which declined in activity during this period. In the mature, sucrose-accumulating mesocarp, the alkaline form I enzyme was the major alpha-galactosidase present. We also observed hydrolysis of raffinose at alkaline conditions in enzyme extracts from other cucurbit sink tissues, as well as from young Coleus blumei leaves. Our results suggest different physiological roles for the alpha-galactosidase forms in the developing cucurbit fruit, and show that the newly discovered enzyme plays a physiologically significant role in photoassimilate partitioning in cucurbit sink tissue.     

Narcissus latent, a virus with filamentous particles and a novel combination of properties

As previously reported, narcissus latent virus (NLV) has flexuous filamentous particles measuring c. 650 nm × 13 nm, is manually transmissible to Nicotiana clevelandii and Tetragonia expansa, and is transmitted by the aphid Myzus persicae following brief acquisition access periods. In contrast to previous reports the virus particle protein has an apparent mol. wt of c. 45 kD. Moreover, infected cells in N. clevelandii leaves contain cytoplasmic inclusion bodies resembling those of potyviruses. In vitro translation of NLV RNA produced only one major product (mol. wt c. 25 kD) which was not precipitated by antisera to virus particle protein or to cytoplasmic inclusion protein. Antisera to 12 potyviruses and nine carlaviruses failed to react with sap containing NLV particles. Similarly antiserum to NLV particles did not react with particles of seven potyviruses or four carlaviruses. A weak reaction was detected between NLV particles and antiserum to particles of maclura mosaic virus (MMV), a virus which resembles NLV in particle morphology and particle-protein size, and in inducing pinwheel inclusions. The cytoplasmic inclusion proteins (CIPs) of NLV, MMV and from narcissus plants with yellow stripe symptoms were serologically inter-related. These proteins were also serologically related to, and had mol. wt similar to, the CIP of members of the potyvirus group. Particles with the size and antigenic specificity of those of NLV were found consistently in narcissus plants with yellow stripe disease. Narcissus latent and narcissus yellow stripe viruses therefore seem to be synonymous and, together with MMV, have properties distinct from those of any previously described virus group.     

Molecular Characterization of a Cucumber Bulgarian latent virus Isolate from Iran

Cucumber Bulgarian latent virus (CBLV) was first reported from cucumber in Bulgaria in 2003 and has been assigned to the genus Tombusvirus. Ten years after the first and only report of CBLV, an isolate from a cucumber sample collected in Iran was characterized. Its complete genomic sequence was determined and analysed. Except for the coat protein, CBLV shows the highest sequence identities to the isolates of other species of the genus Tombusvirus. However, sequence comparison and phylogenetic analyses based on the coat protein (CP) revealed that CBLV is more closely related to the genus Aureusvirus rather than to the isolates of the genus Tombusvirus. The sequence identities to some aureusviruses are above the species demarcation threshold value, demonstrating that CBLV is an unusual tombusvirus species. This suggests that it is necessary to review the CP threshold value for species demarcation in the genus Aureusvirus. In addition, CBLV has an intermediate genome size compared to other tombus‐ and aureusviruses. Several polyclonal antisera raised against different tombus‐ and aureusviruses were used to assess the serological relation to CBLV. The ELISA results indicate that CBLV is not serologically related to any of those tested.     

Vaccinia virus encapsidates a novel topoisomerase with the properties of a eucaryotic type I enzyme

A DNA topoisomerase has been purified from vaccinia virus cores. The native enzyme is composed of a single subunit of 32,000 daltons. The enzyme relaxes both positively and negatively supercoiled DNA in the absence of an energy cofactor. Enzymatic activity is stimulated by magnesium ions and inhibited by ATP, and during relaxation the topoisomerase changes the linking number of the DNA substrate by steps of one. Trapping of the covalent DNA-enzyme intermediate has been achieved, and analysis of the cleavage of duplex DNA by the enzyme reveals that it makes a single-strand break and forms a covalent bond through the 3'-end of the broken strand. Enzymatic activity and formation of the trapped intermediate are inhibited by the drugs novobiocin, coumermycin A1, and berenil. The virally encapsidated enzyme has novel properties but most closely resembles a eucaryotic type I topoisomerase.     

Saffold virus, a novel human Cardiovirus with unknown pathogenicity

Although cardioviruses have been thought to mainly infect rodents, a novel human cardiovirus, designated Saffold virus (SAFV), was identified in 2007. SAFV is grouped with Theiler-like rat virus and Theiler's murine encephalomyelitis virus (TMEV) in the species Theilovirus of the genus Cardiovirus of the family Picornaviridae. Eight genotypes of SAFV have now been identified. SAFV has been isolated from nasal and stool specimens from infants presenting with respiratory and gastrointestinal symptoms as well as from children with nonpolio acute flaccid paralysis; however, the relationship of SAFV to this symptomatology remains unclear. Of note, the virus has also been isolated from the cerebrospinal fluid specimens of patients with aseptic meningitis. This finding is of interest since TMEV is known to cause a multiple sclerosis-like syndrome in mice. The involvement of SAFV in various diseases (e.g., respiratory illness, gastrointestinal illness, neurological diseases, and type I diabetes) is presently under investigation. In order to clarify the pathogenicity of SAFV, additional epidemiological studies are required. Furthermore, identification of the SAFV cellular receptor will help establish an animal model for SAFV infection and help clarify the pathogenesis of SAFV-related diseases. In addition, investigation of the tissue-specific expression of the receptor may facilitate development of a novel picornavirus vector, which could be a useful tool in gene therapy for humans. The study of viral factors involved in viral pathogenicity using a reverse genetics technique will also be important.     

Melon rugose mosaic virus, the cause of a disease of watermelon and sweet melon

A mechanically transmissible virus with isometric particles c. 32 nm in diameter, was isolated from infected watermelons and sweet melons in the People's Democratic Republic of Yemen. Purified virus preparations contained two major sedimenting components with sedimentation coefficients of 61S and 117S. In isopycn ic centrifugation in CsCl the particles formed a single band of buoyant density 1.39 g cm^-3. Preparations of virus particles comprised of a single polypeptide of mol. wt c. 22 000 and ssRNA of mol. wt 2.1 × 10⁶. The virus was serologically related to three of six subgroups of tymoviruses tested. The name melon rugose mosaic virus is proposed for this newly described virus.     

Characterization of a novel endopolygalacturonase from Aspergillus niger with unique kinetic properties

We isolated and characterized a new type of endopolygalacturonase (PG)-encoding gene, pgaD, from Aspergillus niger. The primary structure of PGD differs from that of other A. niger PGs by a 136 amino acid residues long N-terminal extension. Biochemical analysis demonstrated extreme processive behavior of the enzyme on oligomers longer than five galacturonate units. Furthermore, PGD is the only A. niger PG capable of hydrolyzing di-galacturonate. It is tentatively concluded that the enzyme is composed of four subsites. The physiological role of PGD is discussed.     

Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon

Carotenoids are nutritionally important tetraterpenoid pigments that upon oxidative cleavage give rise to apocarotenoid (norisoprene) aroma volatiles. beta-Carotene is the predominant pigment in orange-fleshed melon (Cucumis melo L.) varieties, reaching levels of up to 50 microg/gFW. Pale green and white cultivars have much lower levels (0-10 microg/gFW). In parallel, beta-ionone, the 9,10 cleavage product of beta-carotene, is present (12-33ng/gFW) in orange-fleshed melon varieties that accumulate beta-carotene, and in much lower levels (0-5 ng/gFW) in pale green and white fleshed varieties. A search for a gene putatively responsible for the cleavage of beta-carotene into beta-ionone was carried out in annotated melon fruit EST databases yielding a sequence (CmCCD1) highly similar (84%) to other plant carotenoid cleavage dioxygenase genes. To test its function, the clone was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. We show here that the CmCCD1 gene product cleaves carotenoids at positions 9,10 and 9',10', generating geranylacetone from phytoene; pseudoionone from lycopene; beta-ionone from beta-carotene, as well as alpha-ionone and pseudoionone from delta-carotene. CmCCD1 gene expression is upregulated upon fruit development both in orange, pale-green and white melon varieties, despite the lack of apocarotenoid volatiles in the later. Thus, the accumulation of beta-ionone in melon fruit is probably limited by the availability of carotenoid substrate.     

Development of a novel antimicrobial peptide, AG-30, with angiogenic properties

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an α-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo , we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.     

Characterization of a novel parainfluenza virus,caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus)

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.     

The occurrence, hosts and properties of lilac chlorotic leafspot virus, a newly recognised virus from Syringa vulgaris

Lilac chlorotic leafspot virus (LCLV), a hitherto undescribed virus, was isolated from three of 65 lilacs (Syringa vulgaris) with chlorotic leafspotting symptoms growing in S.E. England. The virus was transmitted readily by sap-inoculation to 21 of 52 species from eight of 20 families, but it was not seed-borne in four hosts or transmitted in the semi-persistent manner by any of four aphid species. The virus was moderately stable in vitro; sap from Chenopodium quinoa was infective after 10 min at 60 but not 65 ^oC, after 8–16 days at 20 ^oC or 25–30 wk at 2 ^oC, and after dilution to 10^-3 but not 10^-4. Up to 180 mg of purified virus per kg leaf tissue were obtained from C. quinoa by clarification of buffered leaf extracts with 8% (v/v) n-butanol, followed by one cycle of differential centrifugation and molecular permeation chromatography on controlled pore glass beads (700 Å, 120–200 mesh). LCLV has fragile flexuous filamentous particles which, when intact, mostly measured c. 12-5 times 1500–1600 nm; the helical substructure (pitch c. 3–7 nm) was clearly visible on some particles mounted in uranyl acetate. The particles sedimented as a single component (sedimentation coefficient 96 S; buoyant density 1–302 g cm^-3) and contained c. 5% nucleic acid and a single polypeptide of mol. wt 27 times 10³. Although these properties place LCLV in the closterovirus group, the virus showed no serological relationship to any of six closteroviruses (beet yellows, beet yellow stunt, carnation necrotic fleck, apple chlorotic leafspot, apple stem grooving and potato virus T) and differed from other recognised or possible members of this group in host range and/or symptoms induced in indicator species. The infrequent occurrence of LCLV in lilac in S.E. England indicates that the virus could probably be eradicated by selecting only virus-free plant material for propagation.     

Occurrence and physico-serological properties of potato virus S (PVS) in Iran

1818 collected samples of potato plant showing virus infection symptoms from 85 fields were tested for PVS infection using DAS-ELISA. Average of infection to this virus varied from 0 to 100%. Least infection was belonging to fields with new introduced varieties. On the other hand native and old introduced cultivars showed heavy infection. In field condition, PVS infected plants didn't show very obvious symptoms, so some infected plants may be missed in field sample collecting. The physical properties of 3 isolates, Avaj, Stanboly and Agria No 15 were determined. TIP 55-60 degrees C, DEP 10(-3) and Liv measured 3-4 days. Ouchterlony agar double diffusion test using SDS was useful for virus detection and precipitation lines didn't show any spur between isolates, although isolates differs slightly in symptomatology. SDS-Page and Western blotting methods used successfully for virus detection and determining and measuring viral protein components.     

Alkaline protease associated with virus particles of a nuclear polyhedrosis virus: assay, purification, and properties

Proteolytic activity was detected within polyhedra of the nuclear polyhedrosis virus of Spodoptera littoralis. The enzyme activity was detected by its ability to degrade the major structural polypeptide of polyhedra (polyhedrin). A quantitative assessment of activity was made by a radioassay technique using ³H-labeled polyhedrin as the substrate. Of the structural components of polyhedra, virus particles showed the greatest specific proteolytic activity. Preparations of purified nucleocapsids were inactive. The virus particle enzyme displayed a temperature optimum for proteolysis of 30 to 40°C and a pH optimum of 9.6. Its activity was inhibited by H²⁺ and Cu²⁺, but not by 2-mercaptoethanol. The enzyme was purified from detergent-treated virus particles by affinity column chromatography, using polyhedrin linked to cyanogen bromide-activated Sepharose. Three major envelope polypeptides (L107, L85, and L71) bound to the column at 4°C, but after incubation at 31°C, polypeptide L71 alone was eluted. The fractions containing this protein exhibited a specific enzyme activity more than 80-fold greater than that present in polyhedra. The possible significance of the alkaline protease, and other proteins with affinity for polyhedrin, is discussed.     

Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice.

Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transformation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.     

Purification and properties of Australian lucerne latent virus, a seed-borne virus having affinities with nepoviruses

An isolate of Australian lucerne latent virus (ALLV) from lucerne in New Zealand was mechanically transmitted to a few herbaceous hosts. It induced diagnostic symptoms in several species of the Chenopodiaceae, but was symptomless in most other hosts including lucerne and Trifolium subterraneum. It was seed transmitted in lucerne. When assayed to Chenopodium quinoa, infective C. quinoa sap lost infectivity after diluting to 10^-4, heating for 10 min at 55°C and storage for 4 days at 4°C. ALLV was purified from infected C. quinoa or pea plants by extracting sap in 0.1 m borate buffer (pH 7) containing 0.2% 2-mercaptoethanol and clarifying with 15% bentonite suspension, high and low speed centrifugation and sucrose density gradient centrifugation. Purified virus preparations contained isometric particles about 25 nm in diameter and sedimented as three virus components with sedimentation coefficients (s_20-w⁰) of 56 S, 128 S and 133 S. The 56 S component appeared to consist of nucleic acid-free protein shells. Polyacrylamide gel electrophoresis of virus preparations showed that ALLV contained a single protein species of mol. wt 55 000 and two RNA species of mol. wt 2.1 × 10⁶ and 2.4 × 10⁶. An antiserum to ALLV had an homologous titre of 1/256 to purified virus but failed to detect ALLV in infective sap of C. quinoa, pea or lucerne. Purified ALLV failed to react to antisera to 28 distinct isometric plant viruses including those to 10 nepoviruses.     

cmv1 is a gate for Cucumber mosaic virus transport from bundle sheath cells to phloem in melon

Cucumber mosaic virus (CMV) has the broadest host range among plant viruses, causing enormous losses in agriculture. In melon, strains of subgroup II are unable to establish a systemic infection in the near‐isogenic line SC12‐1‐99, which carries the recessive resistance gene cmv1 from the accession PI 161375, cultivar ‘Songwhan Charmi’. Strains of subgroup I overcome cmv1 resistance in a manner dependent on the movement protein. We characterized the resistance conferred by cmv1 and established that CMV‐LS (subgroup II) can move from cell to cell up to the veins in the inoculated leaf, but cannot enter the phloem. Immunogold labelling at transmission electron microscopy level showed that CMV‐LS remains restricted to the bundle sheath (BS) cells in the resistant line, and does not invade vascular parenchyma or intermediary cells, whereas, in the susceptible line ‘Piel de Sapo’ (PS), the virus invades all vein cell types. These observations indicate that the resistant allele of cmv1 restricts systemic infection in a virus strain‐ and cell type‐specific manner by acting as an important gatekeeper for virus progression from BS cells to phloem cells. Graft inoculation experiments showed that CMV‐LS cannot move from the infected PS stock into the resistant cmv1 scion, thus suggesting an additional role for cmv1 related to CMV transport within or exit from the phloem. The characterization of this new form of recessive resistance, based on a restriction of virus systemic movement, opens up the possibility to design alternative approaches for breeding strategies in melon.