Improvement of enzymatic stability and intestinal permeability of deuterohemin-peptide conjugates by specific multi-site N-methylation.

The deuterohemin-peptide conjugate, DhHP-6 (Dh-ß-AHTVEK-NH(2)), is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species (ROS). In this study, specific multi-site N-methylated derivatives of DhHP-6 were designed and synthesized to improve metabolic stability and intestinal absorption, which are important factors for oral delivery of therapeutic peptides and proteins. The DhHP-6 derivatives were tested for (1) scavenging potential of hydrogen peroxide (H(2)O(2)); (2) permeability across Caco-2 cell monolayers and everted gut sacs; and (3) enzymatic stability in serum and intestinal homogenate. The results indicated that the activities of the DhHP-6 derivatives were not influenced by N-methylation, and that tri-N-methylation of DhHP-6 could significantly increase intestinal flux, resulting in a two- to threefold higher apparent permeability coefficient. In addition, molecules with N-methylation at selected sites (e.g., Glu residue) showed high resistance against proteolytic degradation in both diluted serum and intestinal preparation, with 50- to 140-fold higher half-life values. These findings suggest that the DhHP-6 derivatives with appropriate N-methylation could retain activity levels equivalent to that of the parent peptide, while showing enhanced intestinal permeability and stability against enzymatic degradation. The tri-N-methylated peptide Dh-ß-AH(Me)T(Me)V(Me)EK-NH(2) derived from this study may be developed as a promising candidate for oral administration.

A novel disulfide-stabilized single-chain variable antibody fragment against rabies virus G protein with enhanced in vivo neutralizing potency.

Rabies is a fatal infectious disease requiring efficient protection provided by post-exposure prophylaxis (PEP) with rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv) is a small engineered antigen binding protein derived from antibody variable heavy (V(H)) and light (V(L)) chains. This novel antibody format may potentially replace the current application of RIG to detect and neutralize rabies virus (RV). However, the broad use of scFvs is confined by their generally low stability. In this study, a scFv (FV57) was constructed based on the monoclonal antibody, MAB57, against RV. To enhance its stability and neutralizing potency, a disulfide-stabilized scFv, ds-FV57, was also derived by introduction of cysteines at V(H)44 and V(L)100. Furthermore, the cysteine at V(L)85 of ds-FV57 was mutated to serine to construct ds-FV57(VL85Ser) in order to avoid potential mis-formed disulfide bonds which would alter the affinity of the scFv. The stability and activity of all three proteins expressed in Escherichia coli were evaluated. All of the constructed scFvs could provide efficient protection against RV infection both in vivo and in vitro. However, the stability of ds-FV57(VL85Ser) was notably improved, and its in vitro neutralizing potency against RV infection was enhanced. Our findings from these stabilization modifications support the feasibility of developing scFvs for PEP treatment of rabies.

Identification of binding epitope for anti-rabies virus glycoprotein single-chain Fv fragment FV57.

Single-chain Fv fragment (scFv) of anti-rabies glycoprotein (G protein) has been recommended as a new agent for detecting and neutralizing lethal rabies virus. In this study, we constructed scFv that corresponded to the FV fragment of CR57, a monoclonal antibody against rabies virus, and called it FV57. Despite its virus neutralization activity, FV57 may or may not recognize the same epitope as that recognized by CR57. To resolve this issue, the binding epitope of rabies virus G protein recognized by FV57 was identified. A recombinant rabies virus G protein fragment (RVG179; residues 179-281) comprising several epitopes was expressed in E.coli, purified, and the specificity of its binding with FV57 was determined. In addition, a peptide (abbreviated as EP, residues 224-236) comprising the known epitope of G protein to which CR57 binds was synthesized and the potency of its binding with FV57 was also determined. The results showed that FV57 could specifically bind to RVG179 and EP. Competitive ELISA experiments indicated that RVG179 and EP were able to compete with the rabies virus G protein for binding with FV57. Since no other epitope within residues 224- 236 has been reported, except for the epitope to which CR57 binds (residues 226-231), the epitope recognized by FV57 was the same as its intact antibody CR57. This demonstrated that the complementarity-determining regions (CDRs) of the heavy and light chains of FV57 have folded into the correct conformation as those of CR57.

Distribution of human papillomavirus genotypes in archival cervical lesions in eastern inner Mongolian autonomous region, China.

INTRODUCTION: Human papillomavirus (HPV) is the main etiologic factor for cervical cancer (CC). To investigate the prevalence of HPV types in archival CC and its precursors collected form Tongliao area, which is located in the east of Inner Mongolian autonomous region, China, and compare the genotype distribution of HPV in cervical lesions between Han Chinese and Mongolian. METHODS: The infections of HPV in a total of 175 cases of formalin-fixed, paraffin-embedded samples, including 71 low-grade squamous intraepithelial lesions (LSIL), 27 high-grade squamous intraepithelial lesions (HSIL), and 77 CC were detected by the combination of consensus primers nested polymerase chain reaction (PCR) and type-specific primers nested PCR. RESULTS: Overall, HPV prevalence was 93.5% in CC, 92.6% in HSIL, and 63.4% in LSIL. Human papillomavirus 16 was the most predominant HPV type in all cervical lesions, detected in 83.1% of CC, 77.8% of HSIL, and 33.8% of LSIL. Human papillomavirus 45 was the second most predominant HPV type in CC (16.9%) and HSIL (11.1%). Human papillomavirus 33 was the second most predominant HPV type in LSIL (8.5%). Human papillomavirus 18, equal with HPV 45, was the second most common type in Mongolian CC (15.6%), whereas in Han Chinese specimens, no HPV 18 was found. CONCLUSIONS: The prevalence of HPV 45 in CC and HSIL in Tongliao area were relatively higher than other regions of China. Comparing the distribution of HPV types in Han Chinese and Mongolian, the prevalence of HPV 18 in CC from Mongolian was significantly higher than that in Han Chinese.

Profile of physical status and gene variation of human papillomavirus 58 genome in cervical cancer.

Epidemiological studies have shown that human papillomavirus 58 (HPV 58) is found at a relatively high frequency in east Asia and some regions of Central and South America. To investigate the physical status of HPV 58 and analyse sequence variations of HPV 58 in cervical cancer patients, the HPV 58 genome in 37 HPV 58-positive cervical cancer specimens collected from China were investigated by a mapping analysis based on nested PCR and nucleotide sequencing. A pure integrated genome was found in 78.4 % (29/37) of specimens, which is much higher than that found in previous studies. Multiple disruptions were first found among the integrated HPV 58 genomes in 51.7 % (15/29) of specimens. Among the 7824 bp of the HPV 58 genome, 119 (1.52 %) nucleotide positions were found to be variable, and 45 of them lead to amino acid changes. Phylogenetic analyses, based on partial L1 sequences of 14 variants isolated in previous studies and this study, show that two main groups were observed in HPV 58 variants, the prototype or prototype-like group and the non-prototype-like group.

Evaluation of high-risk human papillomaviruses type distribution in cervical cancer in Sichuan province of China.

BACKGROUND: Infection with high-risk human papillomavirus is an important factor associated with cervical cancer, and the distribution of HPV types varies greatly worldwide. Determination of type-specific HPV prevalence constitutes an important step towards the development of vaccines for the prevention of cervical cancer. METHODS: The human papillomavirus (HPV) genotypes in 190 cervical cancer specimens taken from the Sichuan province, the most populous province of Southwest China, were detected by a combination of MY09/11 consensus primers PCR (MY09/11 PCR), type-specific primers one-step PCR (One-step TS PCR) and E6/E7 gene type-specific primers nested PCR (Nested TS PCR). The prevalence and distribution of HPV in patients with cervical cancer, especially for HPV types 16, 18, 52, 58 and 59, suspected to be most common in certain parts of China, was investigated. RESULTS: The HPV infection rates detected by MY09/11 PCR, One-step TS PCR and Nested TS PCR were 159 (83.7%), 145 (76.3%) and 172 (90.5%), respectively. The overall HPV prevalence was 93.2% (177/190). The positive specimens for HPV16, 18, 52, 58 and 59 detected by One-step TS-PCR were 111 (58.4%), 14 (7.4%), 6 (3.2%), 13 (6.8%) and 4 (2.1%), respectively. By Nested TS-PCR analysis, the detection rates of HPV16, 52, 58 and 59 were increased to 140 (73.7%), 30 (15.8%), 37 (19.5%) and 25 (13.2%), while only 4 (2.1%) additional specimens were found to be infected with HPV18. CONCLUSION: Our data demonstrate that, besides HPV 16, which was found to be the most prevalent type, HPV types 58, 52 and 59 are more prevalent than HPV18 in women with cervical cancer in the Sichuan area of China.

[Studies on antigencity of human immunodeficiency virus type 1 (HIV-1) external glycoprotein as well as its expression in Pichia pastoris].

Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.

HPV prevalence, E6 sequence variation and physical state of HPV16 isolates from patients with cervical cancer in Sichuan, China.

OBJECTIVES: Infection with high-risk human papillomavirus (hr-HPV) is an important factor associated with cervical cancer. The genetic mutation of HPV16 E6 and integration of HPV16 DNA in the cervical carcinoma tissues are considered important genetic changes in cervical lesion progression. But the studies of hr-HPV epidemiology are relatively less in the area of Sichuan, China. Therefore, we investigated the prevalence of 9 high-risk subtypes and analyzed the genetic mutation characteristic of HPV16 E6 and physical state of HPV16 DNA. METHODS: The fragments of L1 and E6 genes were amplified by PCR or nested PCR and then directly sequenced. Further, the multiplex PCR for HPV16 E2 and E6 genes was performed for detection of integration. RESULTS: HPV16, 58 and 18 were prominent, accounting for 78.6%, 20.0% and 9.7%, respectively in 145 isolates. E6 variants revealed that the European (EP) prototype and East Asia (EA) strain were 26 (23.0%) and 34 (30.1%), respectively. Furthermore, there were 14 base substitutions in E6 regions of the study group, of which 12 resulted in amino acid changes and the rest was silent mutation. Significantly, the 240G substitution exactly located the P53 degradation site. Overall, 8 of 114 (7.0%) isolates only contained integrated HPV16 DNA, 43 (37.7%) only contained episomal DNA and 63 (55.3%) contained both integrated and episomal DNA. The proportion of disruption of an intact E2 gene in the patients with cervical cancer is much lower than that in the previous studies. CONCLUSIONS: HPV16, 58 and 18 were mainly prevailing subtypes in patients with cervical cancer from Sichuan areas, China and EP/EA strains were predominant in these areas. Some mutations of E6 gene, which lead to the amino acid changes, may be more potentially carcinogenic and the proportion of disruption of an intact E2 gene is much lower.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1alpha subunit.

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B1 therapy. T231A has very low activity and a greatly elevated Km for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.