Biomedical subjects

Xiaofeng Guo

Publications and source records attributed to Xiaofeng Guo.

11 recordsLinked to original sources

PubMed2006

Physiological analysis of Methylobacterium extorquens AM1 grown in continuous and batch cultures.

Chemostat cultures of Methylobacterium extorquens AM1 grown on methanol or succinate at a range of dilution rates were compared to batch cultures in terms of enzyme levels, poly-beta-hydroxybutyrate content, and intracellular concentrations of adenine and pyridine nucleotides. In both chemostat and batch cultures, enzymes specific to C1 metabolism were up-regulated during growth on methanol and down-regulated during growth on succinate, polyhydroxybutyrate levels were higher on succinate, intracellular ATP levels and the energy charge were higher during growth on methanol, while the pools of reducing equivalents were higher during growth on succinate. For most of the tested parameters, little alteration occurred in response to growth rate. Overall, we conclude that the chemostat cultivation conditions developed in this study roughly mimic the growth in batch cultures, but provide a better control over the culturing conditions and a better data reproducibility, which are important for integrative functional studies. This study provides baseline data for future work using chemostat cultures, defining key similarities and differences in the physiology compared to existing batch culture data.

Adenine Nucleotides↗

PubMed2006

Association of RNA helicase a with human immunodeficiency virus type 1 particles.

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription.

Autoantigens↗

PubMed2005

The R362A mutation at the C-terminus of CA inhibits packaging of human immunodeficiency virus type 1 RNA.

The capsid (CA) sequence of human immunodeficiency virus type 1 (HIV-1) Gag protein consists of two independently folded domains named the N-terminal domain (NTD) and C-terminal domain (CTD) that are connected by a flexible linker. Most of the CTD sequence adopts rigid structure except for the last 11 amino acids (positions 354 to 364) that are disordered even in the context of the downstream SP1 and nucleocapsid (NC) sequence. Although disordered, this short peptide region plays a crucial role in HIV-1 replication. In this study, we identified three second-site mutations within Gag named A238T, G358S, and N373K that rescued a deleterious mutation R362A located at the C-terminus of CA. A238T is located within the NTD of CA, G358S and N373K are positioned proximal to R362A. One of the mechanisms underlying this compensation event is correction of reduced packaging of viral RNA into the R362A mutated viruses, as shown by the results of RNase protection assays, native Northern blots experiments as well as filter-binding assays. These data suggest that one potential function for the C-terminal disordered sequence of CA in HIV-1 replication is to regulate HIV-1 RNA packaging.

Amino Acid Sequence↗

PubMed2005

Opposing effects of the M368A point mutation and deletion of the SP1 region on membrane binding of human immunodeficiency virus type 1 Gag.

The Gag protein of human immunodeficiency virus type 1 (HIV-1) contains a 14-amino-acid region termed SP1 that is located between the capsid (CA) and nucleocapsid (NC) domains. It has been previously observed that either a M368A substitution within SP1 or the DeltaSP1 deletion impaired virus production. In this study, we further showed that the M368A point mutation, but not the DeltaSP1 deletion, severely diminished the levels of membrane-associated Gag proteins. This membrane binding defect associated with M368A was corrected either by changing NC to the leucine zipper (LZ) motif derived from the yeast transcription factor GCN4 or by a L364A second-site mutation in the context of the first four residues of SP1. Yet, neither the L364A mutation nor the LZ substitution restored wild type levels of particle production to the M368A Gag. These results suggest that SP1 affects both Gag-membrane binding and the subsequent events of virus assembly such as capsid morphogenesis or virus budding.

Amino Acid Sequence↗

PubMed2005

Mutation of the SP1 sequence impairs both multimerization and membrane-binding activities of human immunodeficiency virus type 1 Gag.

The Gag protein of human immunodeficiency virus type 1 contains a 14-amino-acid region, termed SP1, between the capsid and downstream nucleocapsid sequences. Although SP1 is known to be indispensable for virus production, the mechanisms involved are mostly unclear. In this study, we demonstrate that an M368A mutation within SP1 severely diminished the ability of Gag to associate with cellular membranes. Although wild-type levels of membrane binding were restored to the M368A Gag by a second-site L20K mutation within matrix, the resultant Gag mutant L20K-M368A remained defective in virus production. This latter deficit was partially consequent to the binding of L20K-M368A Gag to nonraft membranes as opposed to raft association seen for wild-type Gag. Further analysis revealed that the majority of membrane-bound M368A Gag proteins were small oligomers, indicating a multimerization defect. In support of this observation, purified recombinant Gag derivatives containing the M368A mutation formed much lower amounts of high-molecular-weight complexes that were pelletable at 21,000 x g than did wild-type Gag. Based on the myristyl switch model, we propose that the M368A mutation inhibits Gag multimerization and, as a result, restricts the binding of Gag to cellular membranes.

Animals↗

PubMed2004

Important role for the CA-NC spacer region in the assembly of bovine immunodeficiency virus Gag protein.

Lentiviral Gag proteins contain a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain. This short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (HIV-1). We have now extended this finding to the CA-NC spacer motif within the Gag protein of bovine immunodeficiency virus (BIV). Mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to the severely disrupted association of the mutated Gag with the plasma membrane, as shown by the results of membrane flotation assays and confocal microscopy. Detailed mutagenesis analysis of the BIV CA-NC spacer region for virus assembly determinants led to the identification of two key residues, L368 and M372, which are separated by three amino acids, 369-VAA-371. Incidentally, the same two residues are present within the HIV-1 CA-NC spacer region at positions 364 and 368 and have also been shown to be crucial for HIV-1 assembly. Regardless of this conservation between these two viruses, the BIV CA-NC spacer could not be replaced by its HIV-1 counterpart without decreasing virus production, as opposed to its successful replacement by the CA-NC spacer sequences from the nonprimate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus and visna virus, with the sequence from FIV showing the highest effectiveness in this regard. Taken together, these data suggest a pivotal role for the CA-NC spacer region in the assembly of BIV Gag; however, the mechanism involved therein may differ from that for the HIV-1 CA-NC spacer.

Amino Acid Motifs↗

PubMed2004

[Long-term follow-up observation of clinical therapy for laryngeal carcinoma recurrence and cervical metastasis].

OBJECTIVE: To observe the long-term effects of clinical treatment for laryngeal carcinoma recurrence and cervical metastasis, and discuss the operative therapy method for laryngeal carcinoma recurrence and cervical metastasis. METHOD: Retrospective analysis the 70 patients with laryngeal carcinoma recurrence and cervical metastasis who was cured in our hospital from 1990 to 1998, including 31 local recurrence, 15 stomal recurrence and 24 cervical lymph node metastasis. RESULT: Sixty-four cases were observed over 3 years and 41 cases were studied over 5 years. Twenty-eight patients who received the second-time operation survived over 5 years. In 31 patients with local recurrence, 19 patients received total laryngectomy and 15 patients of them are now living, but 12 patients who did not accept surgical therapy were died in 1-3 years separately; 9 patients with stomal recurrence received the second-time operation and 5 of them lived 5 years longer, however, the 6 patients with stomal recurrence who did not accept the second-time operation all died in 18 months; In 24 patients with cervical lymph node metastasis, 15 patients received the neck dissection and 8 of them are now alive, but the rest patients who did not accept the operation died in 2 years. CONCLUSION: Regular re-examination and systemic clinical follow-up studying are very important for early detection and early treatment in laryngeal carcinoma recurrence and cervical metastasis; Second-time surgical operation is an effective therapeutic method for laryngeal carcinoma recurrence and cervical metastasis; Radiotherapy and chemotherapy play a limited role in clinical therapy for laryngeal carcinoma recurrence and cervical metastasis.

Adult↗

PubMed2003

Suppression of an intrinsic strand transfer activity of HIV-1 Tat protein by its second-exon sequences.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) has been shown to restrict premature reverse transcription at late stages of virus infection and to thus ensure the integrity of the viral RNA genome for packaging. To gain further insights into the roles of Tat in HIV-1 reverse transcription, we have assessed its effects on the first-strand transfer during the synthesis of minus-strand DNA through use of a reconstituted cell-free system. The results demonstrated that a form of Tat, containing only the first exon (Tat72), was able to enhance the first-strand transfer as efficiently as did the viral nucleocapsid protein. Coincidentally, this form of Tat was unable to inhibit the production of minus-strand strong-stop DNA. Further studies with various mutated forms of Tat showed that its Cys-rich region, rather than the core and Arg-rich domains, was essential for this strand transfer activity. Moreover, this activity of Tat is largely independent of the TAR RNA structure. Although full-length Tat protein (Tat86) was also able to promote strand transfer, this activity was limited by a strong overall inhibition of reverse transcription because of the presence of the second Tat exon. Other nucleic-acid-binding proteins (e.g., single-strand DNA-binding protein) were employed as negative controls and were unable to promote strand transfer in these assays. We propose that Tat possesses nucleic acid chaperone activity and can promote the first-strand transfer during HIV-1 reverse transcription; however, these activities are restricted by the second Tat exon, and the roles of these Tat activities in viral replication remain to be elucidated.

Base Sequence↗

PubMed2002

The Tat protein of human immunodeficiency virus type 1 (HIV-1) can promote placement of tRNA primer onto viral RNA and suppress later DNA polymerization in HIV-1 reverse transcription.

Human immunodeficiency virus type-1 Tat has been proposed to play a role in the regulation of reverse transcription. We previously demonstrated that wild-type Tat can augment viral infectivity by suppressing the reverse transcriptase (RT) reaction at late stages of the viral life cycle in order to prevent the premature synthesis of potentially deleterious viral DNA products. Here we have performed a detailed analysis of the cell-free reverse transcription reaction to elucidate the mechanism(s) whereby Tat can affect this process. Our results show that Tat can suppress nonspecific DNA elongation while moderately affecting the specific initiation stage of reverse transcription. In addition, Tat has an RNA-annealing activity and can promote the placement of tRNA onto viral RNA. This points to a functional homology between Tat and the viral nucleocapsid (NC) protein that is known to be directly involved in this process. Experiments using a series of mutant Tat proteins revealed that the cysteine-rich and core domains of Tat are responsible for suppression of DNA elongation, while each of the cysteine-rich, core, and basic domains, as well as a glutamine-rich region in the C-terminal domain, are important for the placement of tRNA onto the viral RNA genome. These results suggest that Tat can play at least two different roles in the RT reaction, i.e., suppression of DNA polymerization and placement of tRNA onto viral RNA. We believe that the first of these activities of Tat may contribute to the overall efficiency of reverse transcription of the viral genome during a new round of infection as well as to enhanced production of infectious viral particles. We hypothesize that the second activity, illustrating functional homology between Tat and NC, suggests a potential role for NC in the displacement of Tat during viral maturation.

DNA Primers↗

PubMed2002

[E-cadherin gene mutation in recurrent and metastatic carcinoma of larynx].

OBJECTIVE: To evaluate the role of E-cadherin gene in the process of recurrent and metastasis of laryngeal cancer and to investigate the metastatic mechanism of laryngeal cancer. METHOD: Exploration of exon 5 and exon 7 of the E-cadherin gene by polymerase chain reaction (PCR), SSCP in the laryngeal cancer. RESULT: In normal laryngeal tissues, there was no E-cadherin gene mutation. E-cadherin gene mutation occured only in the primary carcinoma with cervical metastasis and recurrent laryngeal carcinoma. The mutation rate were 67.71% and 28.57% respectively. E-cadherin gene mutation occured more frequently in advanced carcinomas than that in early ones. CONCLUSION: E-cadherin gene mutation with its functional suppression was one of the molecular mechanisms of the laryngeal metastasis. It is suggested that E-cadherin may be a valuable prognostic marker for the carcinoma of larynx.

Cadherins↗

PubMedDate not supplied

Some notes on 2-D graphical representation of DNA sequence.

Some 2-D and 3-D graphical representations of DNA sequences have been given by Nandy, Leong and Mogenthaler, and Randic et al., which give visual characterizations of DNA sequences. In this paper, we presented a novel graphical representation of DNA sequences by taking four special vectors in 2-D Cartesian coordinate system to represent the four nucleic acid bases in DNA sequences, so that a DNA sequence is denoted on a plane by a directed walk. It is shown that the new graphical representation of DNA sequences has lower or nondegeneracy.

DNA↗