Antibacterial activity and antibacterial mechanism of flavaspidic acid BB against Staphylococcus haemelyticus.

BACKGROUND: Staphylococcus haemolyticus (S. haemolyticus) is the main etiological factor in skin and soft tissue infections (SSTI). S. haemolyticus infections are an important concern worldwide, especially with the associated biofilms and drug resistance. Herein, we investigated the inhibitory effect of Flavaspidic acid BB obtained from plant extractions on clinical S. haemolyticus strains and their biofilms. Moreover, we predicted its ability to bind to the protein-binding site by molecular simulation. Since the combination of Hsp70 and RNase P synthase after molecular simulation with flavaspidic acid BB is relatively stable, enzyme-linked immunosorbent assay (ELISA) was used to investigate Hsp70 and RNase P synthase to verify the potential antimicrobial targets of flavaspidic acid BB. RESULTS: The minimum inhibitory concentrations (MIC) of flavaspidic acid BB on 16 clinical strains of S. haemolyticus was 5 ~ 480 µg/mL, and BB had a slightly higher inhibitory effect on the biofilm than MUP. The inhibitory effect of flavaspidic acid BB on biofilm formation was better with an increase in the concentration of BB. Molecular simulation verified its ability to bind to the protein-binding site. The combination of ELISA kits showed that flavaspidic acid BB promoted the activity of Hsp70 and inhibited the activity of RNase P, revealing that flavaspidic acid BB could effectively inhibit the utilization and re-synthesis of protein and tRNA synthesis, thus inhibiting bacterial growth and biofilm formation to a certain extent. CONCLUSIONS: This study could potentially provide a new prospect for the development of flavaspidic acid BB as an antibacterial agent for resistant strains.

[Determination of the in vitro antiviral activity of an engineered M1GS ribozyme that targets to the core gene of hepatitis C virus].

OBJECTIVE: Hepatitis C virus (HCV) is one of the major pathogens that lead to viral hepatitis. At present, Interferon treatment in combination with ribavirin is the first line clinical therapeutic approach. However, the responses are usually poor and the viral infection reoccurs. Therefore, exploring new antiviral agents and therapies is under urgent needs. METHODS: The sequence and structure of the core coding region of HCV genome were analyzed through the two computer software, DNAMAN and RNA Structure. The cytosine 52 nt downstream of the AUG initiation triplet was identified as the optimal target cleavage site. Based on the flanking sequence of this assumed cleavage site, a guide sequence (GS) was designed and covalently linked to the 3 prime terminus of the M1 RNA, which is catalytic subunit of the RNase P derived from Escherichia coli using PCR. We named this new targeting ribozyme M1GS-HCV/C52 and it antiviral activities were analyzed in cultured cells. RESULTS: In the in vitro cleavage assay, M1GS-HCV/C52 ribozyme could effectively cleave the HCV target RNA into two fragments at the specific cleavage site. Moreover, comparing to the blank control, this engineered M1GS ribozyme could reduce the core protein expression of more than 80% in the HCV-infected host cell and lead to a 1500-fold reduction of HCV RNA copies in the culture supernatant. An another M1GS ribozyme, M1GS-HCV/C52*, which has the same guide sequence but does not contain a 24nt-long bridge sequence, did not exhibit apparent inhibition for the expression of HCV core gene and viral proliferation in our paralleled assay. CONCLUSION: We successfully constructed an M1GS ribozyme showing affective and specific cleavage of target viral RNA. Further results showed that the engineering ribozyme had notably antiviral activity in cultured cells, thus provided a new promising approach for clinical anti-HCV therapeutic strategy.

Targeting mRNAs by engineered sequence-specific RNase P ribozymes.

The methods of using engineered RNase P catalytic RNA (termed as M1GS RNA) for in vitro and in vivo in trans-cleavage of target viral mRNA are described in this chapter. Detailed information is focused on (1) mapping accessible regions of target viral mRNA in infected cells, (2) generation and in vitro cleavage assay of the customized M1GS ribozyme, (3) stable expression of M1GS RNAs and evaluation of its antiviral activity in cultured cells. Using these methods, we have constructed functional M1GS ribozyme that can cleave an overlapping region of the mRNAs coding for the human cytomegalovirus (HCMV) capsid scaffolding protein (CSP) and assemblin in vitro. Further study has demonstrated that, in cultured human cells expressing the functional M1GS ribozyme and infected with HCMV, more than 85% reduction in the expression of CSP and assemblin and a 4,000-fold reduction in viral growth were achieved. Our study provided the direct evidence that the customized M1GS ribozyme can be used as an effective gene-targeting agent for in trans-cleavage of viral genes and inhibition of viral growth in cultured cells.

[Experimental study on phenotypic conversion of clinical chloromycetin-resistant strains of E. coli to drug-sensitive strains by using EGS technique in vitro].

OBJECTIVE: To explore the possibility of phenotypic conversion of clinical chloromycetin (Cm)-resistant isolates of E.coli to drug-sensitive ones with external guide sequences (EGS) in vitro. METHODS: Recombinant EGS plasmids directed against Cm acetyl transferase (cat) and containing kanamycin (Km) drug-resistance gene and control plasmids only containing kanamycin-resistance gene without EGS were constructed. By using CaCl(2) method, the recombinant plasmids were introduced into the clinically isolated Cm-resistant E.coli strains. Extraction of plasmids and PCR were applied to identify the EGS positive clones; The growth rate in liquid broth culture of Cm-resistant bacteria after EGS containing plasmid transformation was determined by spectrophotometer A(600). Drug sensitivity was tested in solid culture by using KB method. RESULTS: Transformation studies were carried out on 16 clinically isolated Cm-resistant E.coli strains with pEGFP-C1-EGS + cat1 + cat2 recombinant plasmid. Transformants were screened on LB-agar plates containing Km after transformation using EGS. In 4 tested strains of them, transformants with specific EGS plasmid showed growth inhibition when grown in liquid broth culture containing 100 approximately 200 micro g/ml of Cm. They were sensitive to Cm on LB-agar plates containing 100 approximately 200 micro g/ml of Cm in drug-sensitivity test. Extraction of plasmids showed the existence of EGS bands. PCR amplified products of EGS. The above facts indicated that the 4 strains out of the 16 clinical isolates had been converted to drug-sensitive phenotype, and Cm-resistant clinically isolated E. coli resumed sensitivity to Cm. CONCLUSION: EGS has the capability of converting the phenotype of clinical drug-resistant isolates to drug sensitivity.

Effective inhibition of HIV-1 replication in cultured cells by external guide sequences and ribonuclease P.

We examined the suppressive effect of HIV-1 RNA gene cleavage on HIV-1 expression, using the catalytic RNA subunit RNase P and the 3'-half tRNA(Try) [external guide sequence (EGS)] in cultured cells. HIV-1 expression was inhibited by the tRNA(met)-EGS-U5 and U6-EGS-U5 from the tRNA(met) and U6 promoters, respectively. There was no difference in the inhibitory effects on HIV-1 expression between the tRNA(met) and U6 promoters.

RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins.

By linking a guide sequence to the catalytic RNA subunit of RNase P (M1 RNA), we constructed a functional ribozyme (M1GS RNA) that targets the overlapping mRNA region of two human cytomegalovirus (HCMV) capsid proteins, the capsid scaffolding protein (CSP) and assemblin, which are essential for viral capsid formation. The ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, a reduction of >85% in the expression of CSP and assemblin and a reduction of 4000-fold in viral growth were observed in the HCMV-infected cells that expressed the functional ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in virus-infected cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. Characterization of the effects of the ribozyme on the HCMV lytic replication cycle further indicates that the expression of the functional ribozyme specifically inhibits the expression of CSP and assemblin, and consequently blocks viral capsid formation and growth. Our results provide the direct evidence that RNase P ribozymes can be used as an effective gene-targeting agent for antiviral applications, including abolishing HCMV growth by blocking the expression of the virus-encoded capsid proteins.

Effective inhibition of Rta expression and lytic replication of Kaposi's sarcoma-associated herpesvirus by human RNase P.

Ribonuclease P (RNase P) complexed with external guide sequence (EGS) represents a nucleic acid-based gene interference approach to knock-down gene expression. Unlike other strategies, such as antisense oligonucleotides, ribozymes, and RNA interference, the RNase P-based technology is unique because a custom-designed EGS molecule can bind to any complementary mRNA sequence and recruit intracellular RNase P for specific degradation of the target mRNA. In this study, we demonstrate that the RNase P-based strategy is effective in blocking gene expression and growth of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), the causative agent of the leading AIDS-associated neoplasms, such as KS and primary-effusion lymphoma. We constructed 2'-O-methyl-modified EGS molecules that target the mRNA encoding KSHV immediate-early transcription activator Rta, and we administered them directly to human primary-effusion lymphoma cells infected with KSHV. A reduction of 90% in Rta expression and a reduction of approximately 150-fold in viral growth were observed in cells treated with a functional EGS. In contrast, a reduction of <10% in the Rta expression and viral growth was found in cells that were either not treated with an EGS or that were treated with a disabled EGS containing mutations that preclude recognition by RNase P. Our study provides direct evidence that EGSs are highly effective in inhibiting KSHV gene expression and growth. Exogenous administration of chemically modified EGSs in inducing RNase P-mediated cleavage represents an approach for inhibiting specific gene expression and for treating human diseases, including KSHV-associated tumors.

RNase P as a tool for disruption of gene expression in maize cells.

RNase P, a ribonucleoprotein responsible for the 5' maturation of precursor tRNAs (ptRNAs) in all organisms, can be enticed to cleave any target mRNA that forms a ptRNA-like structure and sequence-specific complex when bound to an RNA, termed the EGS (external guide sequence). In the present study, F3H (flavanone 3-hydroxylase), a key enzyme in the flavonoid biosynthetic pathway that participates in the formation of red-coloured anthocyanins, was used as a target for RNase P-mediated gene disruption in maize cells. Transient expression of an EGS complementary to the F3H mRNA resulted in suppression of F3H to 29% of the control, as indicated by a reduced number of anthocyanin-accumulating cells. This decrease was not observed in experiments where a disabled mutant EGS was expressed. Our results demonstrate the potential of employing plant RNase P, in the presence of an appropriate gene-specific EGS, as a tool for targeted degradation of mRNAs.

RNase P-mediated inhibition of viral growth by exogenous administration of short oligonucleotide external guide sequence.

The use of external guide sequence (EGS) in directing endogenous ribonuclease P (RNase P) for inhibition of viral propagation is described in this chapter, with an emphasis on chemically modified EGSs and their extracellular delivery. Targeting of the mRNA-encoding human cytomegalovirus (HCMV) protease by DNA-based EGSs is presented as an example of how to design chemically modified EGSs for antiviral applications. General information about the EGS-based technology is included, followed by detailed protocols for EGS design, human RNase P purification, in vitro assay of EGS activity, liposome-mediated delivery of chemically modified EGSs and detection of their distribution in cells, and an assay of EGS activity for blocking growth of HCMV in cultured cells.

RNase P ribozyme as an antiviral agent against human cytomegalovirus.

Human cytomegalovirus (HCMV) represents one of the most medically important human viruses and causes a wide spectrum of human diseases, including birth defects and mental retardation in newborns, common opportunistic infections in acquired immunodeficiency syndrome (AIDS) patients (e.g., CMV-associated retinitis and pneumonia), and possibly cardiovascular diseases such as atherosclerosis. This chapter describes the utilization of RNase P ribozyme-specifically, M1GS ribozyme, as a gene-targeting agent for blocking HCMV gene expression and growth. The target for the RNase P ribozyme is the overlapping region of the mRNAs that code for HCMV major transcription factors IE1 and IE2, which are essential for viral gene expression and replication. The methods described in this chapter focus primarily on i) construction of the retroviral vector for expression of M1GS ribozymes in cultured cells, ii) generation of stable cell lines expressing ribozymes, iii) determination of the expression of M1GS RNAs in human cells, and iv) evaluation of the efficacy of ribozymes in inhibiting HCMV IE1/IE2 expression and viral growth. Using these methods, we successfully constructed M1GS RNAs against the IE1/IE2 mRNA sequence and recently showed that a reduction of up to 150- to 3000-fold in HCMV growth is found in cells that express the ribozymes.

[The biological activity of MHC classII transactivator ribonuclease P: a novel approach for hepatic transplantation rejection].

OBJECTIVE: This paper studied the effect of RNaseP against CIITA on repressing class II MHC (MHCII) expression. METHODS: It was constructed that M1-RNA with guide sequences (GS), recognizing the 629 site of CIITA (M1-629-GS), by PCR from pTK117 plasmid, then was cloned into psNAV (psNAV-M1-629-GS). CIITA target gene was obtained from Raji cell by RT-PCR, and then inserted into pGEM-7zf (+) (pGEM-800). psNAV-M1-629-GS and pGEM-800 were transcribed and then mixed up and incubated in vitro. Stable transfectants of hepatocyte with psNAV-M1-629-GS by nanometer were tested for MHCII induction by recombinant human interferon-gamma (IFN-gamma). mRNA abundance of CIITA was measured by RT-PCR. RESULTS: It showed that M1-629-GS could exclusively cleave pGEM-800 that formed a base pair with the GS. When induced with IFN-gamma, the expression of HLA-DR, -DP, -DQ on psNAV-M1-629-GS+ hepatocyte was (1.01+/-0.51)%, (4.37+/-1.28)%, (1.98+/-0.42)% respectively, was down-modulated 90.65%, 89.11% and 65.32% compared with control, while the mRNA content of CIITA reduced significantly (P<0.01). CONCLUSION: M1-629-GS could effectively repress MHCII expressing through cleaving CIITA mRNA. These results provided insight into the future application of it as a new nucleic acid drug against the rejection of hepatic transplantation.

Autoantigenicity of nucleolar complexes.

Autoantibodies targeting nucleolar autoantigens (ANoA) are most frequently found in sera from patients with systemic sclerosis (SSc, also designated scleroderma) or with SSc overlap syndromes. During the last decade an extensive number of nucleolar components have been identified and this allowed a more detailed analysis of the identity of nucleolar autoantigens. This review intends to give an overview of the molecular composition of the major (families of) autoantigenic nucleolar complexes, to provide some insight into their functions and to summarise the data concerning their autoantigenicity.