Preparation of monoclonal antibodies against human ventricular myosin light chain 1 (HVMLC1) for functional studies.

Using purified recombinant human ventricular myosin light chain 1 (HVMLC1) as the antigen, three monoclonal antibodies, designated C8, C9 and B12, were prepared. Immunoblot experiments demonstrated that all monoclonal antibodies could react with the ventricular myosin light chain 1 isolated from different sources, such as human, rat or pig. It was also demonstrated that C8 was directed against the NN part of the N-fragment (amino acid 1-40) of HVMLC1, and both C9 and B12 against the C-fragment (amino acid 99-195). The affinity constants of C8, C9 and B12 were 3.20 x 10(8), 8.600 x 10(7) and 1.770 x 10(8) M(-1), respectively, determined by non-competitive ELISA. The isotype of B12 was determined as IgG2a, whereas the isotype of both C8 and C9 were IgG1. In the presence of C9 or B12, the actin-activated Mg(2+)ATPase activity of myosin was greatly inhibited, but there was almost no effect on the Mg(2+) ATPase activity for C8. B12 and C9 also inhibited the superprecipitation of porcine cardiac native actomyosin (myosin B) and reconstituted actomyosin, but C8 did not. The results indicate that all three monoclonal antibodies could bind the intact myosin molecule, but B12 and C9 might more easily react with epitopes located in the C-fragment of HVMLC1. The inhibitory effects of B12 and C9 on ATPase activity and superprecipitation assays show that light chain 1, particularly the C-fragment domain, is involved in the modulation of the actin-activated Mg(2+) ATPase activity of myosin and, as a consequence, plays an essential role in the interaction of actin and myosin.

[Excision of selectable marker gene from transgenic plant].

Currently used plant transformation systems require selectable marker genes encoding antibiotic or herbicide resistance, along with the gene of interest, to select transformed cells from a large population of mostly untransformed cells. The continued presence of these selectable markers, especially in food crop, is of increasing public concern. The generation of selectable marker-free transgenic plant is one of the new projects in plant biotechnology research. Two techniques, segregation excision and recombination excision, for removal of selectable marker genes are described in this article. The advances in producing selectable marker-free transgenic plants are reviewed too.

Nucleic acid binding activity of pns6 encoded by genome segment 6 of rice ragged stunt oryzavirus.

The ORF of genome segment 6 (S6) of rice ragged stunt oryzavirus (RRSV) Philippines isolate was cloned and sequenced based on the S6 sequence of the Thailand isolate. Pns6, the 71 kD product of S6 expressed in E. coli, was demonstrated to be a viral non-structural protein of RRSV by Western blotting. The gel mobility shift assays showed that Pns6 had nucleic acid binding activity. Pns6 could interact with single- and double-stranded forms of DNA and RNA, showing a preference for single-stranded nucleic acid and a slight preference for RRSV ssRNA over the rice ssRNA, as demonstrated by both competition and displacement assays. The binding of Pns6 to nucleic acids is strong and sequence non-specific. By using five truncated derivatives of Pns6, it was found that the basic region from amino acid 201 to 273 of Pns6 was the unique nucleic acid binding domain. Subcellular fractionation of leaf tissues of RRSV-infected rice plants and subsequent Western blotting had shown that Pns6 accumulated predominately in the cytoplasmic membrane fraction. The possible role of RRSV Pns6 in virus replication and assembly is discussed.

The phosphorylation of NS protein of wheat rosette stunt virus.

The genome of wheat rosette stunt virus (WRSV), a plant rhabdovirus, is a single negative strand RNA. It encodes five viral structural proteins: the glycoprotein (G), the matrix protein (M), the nucleocapsid protein (N), the large protein (L) and the non?structural protein (NS), which was later proved to be a viral structural protein too and existed in a variety of phosphorylation forms in case of vascular stomatitis virus (VSV). In this paper we demonstrated that NS protein of WRSV, either bound with the viral nucleocapsid or expressed in bacteria could be in vitro phosphorylated in presence of viral nucleocapsid. We concluded that the NS protein of WRSV was a phosphorylated protein and it might exist in both phosphorylated and dephosphorylated forms in virions. Our results excluded the possibility that the NS protein could be autophosphorylated. The L protein, the major component of viral RNA dependent RNA polymerase is associated with the protein kinase for phosphorylation of NS protein.

[Self-aggregation of the structural protein encoded by rice ragged stunt Oryzavirus genome segment 8].

Rice ragged stunt oryzavirus (RRSV) is a member of the genus oryzavirus within the family Reoviridae. Its genome consists of ten segments of dsRNA. The functions of all products encoded by these viral genome segments, except one encoded by S9, have not yet been elucidated. In the present study, the ORF of S 8 of RRSV-Philippines isolate was sequenced and expressed in E. coli. The 67 kD product of S8 could be self-cleaved into two fragments with molecular weights of 43 kD and 26 kD. Western blotting indicated that both 67 kD and 43 kD products were major structural proteins of the virus. It was also found that the 67 kD protein could self aggregate into aggregates with higher sedimentation rate in sucrose gradients during centrifugation. Moreover, the self-aggregation process could be accelerated by the complex of S6 product and genome dsRNAs of RRSV. These results suggest that the S8 products, 67 kD or 43 kD, may be the structural components of the viral inner-capsid.

Sequence analysis of F gene of SF02 isolate of goose paramyxovirus and its identification by multiplex RT-PCR.

The complete F gene of SF02 of goose paramyxovirus (GPV) has been cloned and analyzed. The sequence analysis demonstrated that the F gene of SF02 contains 1 662 nt and encodes 553 amino acids, and its cleavage activation site of F gene has the same deduced amino acid sequence, (112)R-R-Q-K-R-F(117), as the velogenic (highly pathogenic) strain of newcastle disease virus. The latter correlated with the virulence of the isolate in biological assays. The F gene of SF02 isolate with the domestic standard velogenic strain NDV, F48E9, shared 86.5% homology in nucleotide and 90.8% homology in amino acid sequences. The SF02 isolate is closer to some NDV strains prevalent in Taiwan and West-European countries in recent years. Based on the F gene sequence a multiplex RT-PCR method has been developed. It could be used for the discrimination of GPV from NDV.

[Effect of trigeminus nerve on facialis-denervated facial muscle atrophy].

In the present study we made out an animal model on rabbit whose trigeminus and facialis nerves were simultaneously or only the latter one was severed. The pathological changes in facial muscle atrophy under different nerve injuries were investigated. The degeneration of contractile proteins of upper lip muscle -- myosin and actin was observed. In addition, we also examined the ultrastructural changes in the muscle atrophy in the two above-mentioned nerve injury cases. We observed that the intact trigeminus nerve could delay and lighten the atrophy of facialis-denervated facial muscle and attenuate the degeneration of myosin and actin, as well as decrease the increment of collagen and maintain the ultrastructure of the thick and thin muscle filaments. These results may provide the possibility of improvement of clinical treatment for facial muscle palsy.

Binding of Human Cardiac Myosin Light Chain 1 to Heavy Chain and Actin.

The cloning and sequence analysis of Chinese human cardiac myosin light chain 1 (CCMLC1) was previously reported. In this paper the cDNA of CCMLC1 was used as template and both of cDNAs of N and C terminal fragments of CCMLC1, each containing 98 amino acid residues, were obtained by PCR. Using the expressed products of both fragments, the binding experiments of two fragments to cardiacmyosin heavy chain of rat, human cardiac actin and to monoclonal antibody raised against CCMLC1, have been performed, respectively, by means of precipitation with GST-Sepharose beads. The results showed that all the heavy chain, actin and monoclonal antibody bound the N terminal fragment of CCMLC1 at different sites. Under experimental conditions, the binding of CCMLC1 with actin could affect the subsequent binding of CCMLC1 to heavy chain in topologically.

The Sequence Analysis and in Vitro Cleavage by Ribozymes of a Putative Early and Late Transcription Gene of the Prawn Baculovirus.

Since 1993 there has been outbreak of an acute lethal disease of the cultivated fleshy prawns (Penaeus chinesis, Osbeck) in China. After a short period of intensive studies we reported firstly the presence of a non-occluded baculovirus in the tissue of diseased prawns. The virus was also named as white spot syndrome baculovirus by some authors. Here, it is reported that, based on sequence analysis, an early and late transcription gene(1 197bp gene)of baculovirus was identified. Two hammerhead ribozymes RZ1 and RZ2 targeting the 54-56 bp and 314-316 bp of the 1 197 bp gene were designed, and in vitro cleavage experiments showed that under proper conditions the target gene could be completely cleaved by those ribozymes together or separately.

The Sequence Analysis and Expression of cDNA of Human Cardiac Myosin Light Chain 1 and the Preparation of Monoclonal Antibody to the Expressed Product.

The nucleotide sequence of cDNA of ventricular myosin light chain 1 of Chinese patients was analyzed. Two remarkable differences in deduced amino acid sequence were found by comparison with amino acid sequence reported previously by Jackowski. The cDNA was expressed in E.coli and the expressed product was used for production of specific polyclonal and monoclonal antibodies. Using both of the poly- and monoclonal antibodies, as well as the expressed product, diagnosis kits for Acute Myocardial infarction was constructed (China patent application number 98 122066.5). Detailed studies on physiological function of cardiac myosin light chain 1 is under way in our lab.