Biomedical subjects

Zhijun Li

Publications and source records attributed to Zhijun Li.

At least 19 recordsLinked to original sources

PubMed2025

Rewiring tumor immunity via zinc finger proteins: a new frontier in cancer immunotherapy.

BACKGROUND: Zinc finger proteins (ZFPs) represent the largest and most structurally diverse family of transcription factors in the human genome. They function through characteristic zinc finger domains that enable specific binding to DNA, RNA, and proteins, playing a central regulatory role in the tumor immune microenvironment. MAIN BODY: This review systematically examines the dual functions of ZFPs in dynamically regulating both innate and adaptive immune responses in cancer. At the innate immunity level, ZFPs precisely control dendritic cell (DC) fate determination, dictate macrophage polarization, balance natural killer (NK) cell activation, mediate myeloid-derived suppressor cell (MDSC) immunosuppressive function, and modulate innate immune sensors and inflammasomes. Within adaptive immunity, ZFPs critically influence T cell effector function and regulate B cell differentiation. Building on these, diverse immunotherapeutic strategies targeting ZFPs are now emerging. These include gene-editing, small molecules and proteolysis-targeting chimeras (PROTACs), synergistic combinations with immune checkpoint blockade, and ZFP-engineered chimeric antigen receptor T (CAR-T) cells. CONCLUSIONS: As pivotal nodes within the tumor immune regulatory network, ZFP-targeting strategies offer novel opportunities to overcome current therapeutic bottlenecks.

Humans↗

PubMed2007

Extremely high alkaline protease from a deep-subsurface bacterium, Alkaliphilus transvaalensis.

A new high-alkaline protease (ALTP) was purified to homogeneity from a culture of the strictly anaerobic and extremely alkaliphilic Alkaliphilus transvaalensis. The molecular mass was 30 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed the maximal caseinolytic activity higher than pH 12.6 in KCl-NaOH buffer at 40 degrees C. Hydrolysis of the oxidized insulin B-chain followed by mass spectrometric analysis of the cleaved products revealed that as many as 24 of the total 29 peptide bonds are hydrolyzed in a block-cutting manner, suggesting that ALTP has a widespread proteolytic functions. Calcium ion had no effect on the activity and stability of ALTP, unlike known subtilisins. The deduced amino acid sequence of the enzyme comprised 279 amino acids plus 97 prepropeptide amino acids. The amino acid sequence of mature ALTP was confirmed by capillary liquid chromatography coupled to tandem mass spectrometry, which was the 93% coverage of the deduced amino acid sequence. The mature enzyme showed moderate homology to subtilisin LD1 from the alkaliphilic Bacillus sp. strain KSM-LD1 with 64% identity, and both enzymes formed a new subcluster at an intermediate position among true subtilisins and high-alkaline proteases in a phylogenetic tree of subtilase family A. ALTP is the first high-alkaline protease reported from a strict anaerobe in this family.

Amino Acid Sequence↗

PubMed2006

Gene expression profiling in embryonic mouse lenses.

PURPOSE: In this study, we used laser capture microdissection (LCM) and microarray hybridization technology to compare the gene expression profiles of mouse embryonic days 10 and 12 lenses (E10 and E12). METHODS: Lens cells of C57/BL6 mouse embryos at E10 and E12 were harvested using the PixCell II LCM System. Total RNA was extracted, amplified, labeled, and hybridized to the 430 2.0 mouse chip (Affymetrix) according to the manufacturer's instructions. Data extracted from the images were analyzed using different software programs. Regulated expression of selected genes was confirmed by real-time PCR (RT-PCR). RESULTS: Analysis of the microarray data from E10 and E12 lenses identified 1,573 genes that showed a two fold or greater change in expression level. Among these 1,573 genes, 956 genes were downregulated and 617 were upregulated in E12 lenses. In addition to the upregulated expression of beta- and gamma-crystallin genes, genes that regulate the cell cycle showed significant changes of gene expression during the E10 (lens pit) to E12 (primary fiber cell induction) time period. Genes involved in insulin-like growth factor (IGF) signaling and Wnt (a family of secreted glycoproteins related to the Drosophila segment polarity gene, wingless, and to the proto-oncogene, int-1) signaling were also differentially regulated. In particular, positive regulators of Wnt signaling were downregulated and negative regulators were upregulated, indicating that modulation of Wnt signaling is important for normal lens morphogenesis. CONCLUSIONS: Our results provide new information about differential regulation of gene expression during early lens development. Analysis of global gene expression profiles in embryonic mouse lenses has allowed us to identify several molecular pathways that are differentially regulated during early lens development.

Animals↗

PubMed2006

Cytotoxic gene therapy for human breast cancer in vitro.

BACKGROUND: Transcription factor PDX-1 is expressed by human pancreatic and breast cancers. Although cytotoxicity of PDX-1-directed RIP-TK/GCV gene therapy to pancreatic cancer cells has been demonstrated, the efficacy of this treatment in breast cancer cells is unknown. The purpose of this study was to determine the expression of PDX-1 and its effect on RIP activation in two human breast cancer cell lines, AU565 and T47D. We also investigated the efficacy of RIP-TK/GCV gene therapy and examined whether exogenous PDX-1 to would enhance its cytotoxic effect. MATERIALS AND METHODS: RT-PCR was used to determine PDX-1 expression. Gene constructs RSVLacZ and RIPLacZ were used for transient transfection and LacZ expression was determined using reporter assays. T47D cells were also transfected with adenoviral vectors. Cells were transfected with RIP-TK and the suboptimal level of GCV was determined for each cell line. Following GCV treatment, cytotoxicity was measured using MTS assays. The effect of exogenous PDX-1 on LacZ expression and RIP-TK cytotoxicity was determined. RESULTS: PDX-1 mRNA was expressed in human breast cancer cells and activated the RIP. Exogenous PDX-1 enhanced LacZ expression in AU565 cells but not in T47D cells. Adenoviral transfection was more efficient in T47D cells than non-viral transfection. RIP-TK treatment was cytotoxic to AU565 and T47D cells and this effect was enhanced by exogenous PDX-1 with both transfection methods. CONCLUSIONS: RIP-TK/GCV therapy is cytotoxic to human breast cancer cells and exogenous PDX-1 enhances cytotoxicity. In vivo studies are necessary to determine the tumor specificity and efficacy of this treatment.

Adenoviridae↗

PubMed2006

A simple approach for protein structure discrimination based on the network pattern of conserved hydrophobic residues.

Evolutionarily conserved hydrophobic residues at the core of protein structures are generally assumed to play a structural role in protein folding and stability. Recent studies have implicated that their importance to protein structures is uneven, with a few of them being crucial and the rest of them being secondary. In this work, we explored the possibility of employing this feature of native structures for discriminating non-native structures from native ones. First, we developed a network tool to quantitatively measure the structural contributions of individual amino acid residues. We systematically applied this method to diverse fold-type sets of native proteins. It was confirmed that this method could grasp the essential structural features of native proteins. Next, we applied it to a number of decoy sets of proteins. The results indicate that such an approach indeed identified non-native structures in most test cases. This finding should be of help for the investigation of the fundamental problem of protein structure prediction.

Binding Sites↗

PubMed2006

Specific targeting of pancreatic islet cells in vivo by insulin-promoter-driven adenoviral conjugated reporter genes.

BACKGROUND: The objective of this study was to determine whether rat insulin promoter (RIP) could, in a mouse model, direct expression of an adenovirus-mediated reporter gene specifically into pancreatic islets via systemic delivery. METHODS: Five hundred and eight base pairs of the RIP DNA sequence were constructed into an adenoviral vector containing a lacZ reporter gene (Adeno-RIP-lacZ). The cytomegalovirus (CMV) promoter was constructed to drive lacZ reporter-gene expression (Adeno-CMV-lacZ) and used as controls. In vitro transient transfection assays were performed to determine levels of reporter-gene expression and compared with that of liposome-mediated plasmid transfection. SCID mice were bred and housed in the barrier BL-4 animal facility. At 2 months of age, the human pancreatic cancer cell PANC-1 was intraperitoneally injected into male mice. Two months after the tumor cell inoculation, mice were injected with 10(7) adenoviral particles via tail veins. After gene delivery, mice were sacrificed at different time points to determine transgene expression levels. Complete necropsies were performed. Morphological alterations were determined using hematoxylin and eosin (H&E) staining, and distribution of the reporter lacZ gene was determined by immunohistochemistry analyses. RESULTS: Adenoviral-driven reporter-gene expression resulted in more than 5 times higher transgene expression compared with conventional plasmid transfections. In Adeno-RIP-lacZ-injected mice, lacZ expression was specifically detected in pancreatic islets. By contrast, in Adeno-CMV-lacZ-injected mice, lacZ gene expression was observed in multiple organs and tissues. Mononuclear cell infiltration and liver cell inflammation were found in Adeno-CMV-lacZ-treated mice. Similar phenomena were observed in islet cells of Adeno-RIP-lacZ-treated mice. A significantly higher level of reporter-gene expression was also found at the edge of in-vivo-inoculated human pancreatic tumors. CONCLUSION: These results demonstrate that RIP-directed reporter-gene expression was found specifically in mouse pancreatic islets and implanted human pancreatic cancer cells. These data thus demonstrate that the combination of an adenoviral vector and a tissue-specific promoter could lead to an enhanced and more specific transgene expression in vivo.

Adenoviridae↗

PubMed2006

Significance of nueroelectrophysiological tests in the early diagnosis of sub-clinical neuropathay with diabetes mellitus.

To evaluate the application of nueroelectrophysiological tests in early diagnosis of sub-clinical neuropathy in diabetes mellitus (DM), The routine nerve conductive velocity (NCV), F-wave and sympathetic skin response (SSR) were detected in 27 patients with diabetes mellitus but without symptoms and signs of lesions of nerve system. Our results showed that 48.1%, 44.4%, 51.9% of the patients were found to have abnormal NCV, F-wave and SSR respectively. The abnormalities were mainly characterized by prolonged latency, reduced velocity and absence of wave-form. There were significant differences between the controls and the DM group (P < 0.05). Both the distal and proximal segments of nerves were affected and the distal lesions took place earlier than proximal ones and the changes in low extremities were more severe than those of upper extremities. F-wave can be used as a sensitive indicator for the early diagnosis of peripheral neuropathy and it can help to detect the subclincial lesions. SSR can be used for the evaluation of functional status of autonomic nerves in DM patients.

Adult↗

PubMed2006

Effect of pH management on brain perfusion during retrograde cerebral perfusion.

This study was undertaken to determine the effects of different pH management strategies during retrograde cerebral perfusion on the relationship between retrograde perfusion pressure and brain tissue perfusion. Six pigs were subjected to an alpha-stat strategy and another 6 to a pH-stat strategy during hypothermic (15 degrees C) retrograde cerebral perfusion at perfusion pressures of 10 to 70 mm Hg, in increments of 10 mm Hg every 20 min. Regional cerebral blood flow was significantly higher in the pH-stat group than in the alpha-stat group. The cerebral blood flow peaked at perfusion pressures of 40-50 mm Hg (18.6% +/- 10.8% in the pH-stat group vs. 3.6% +/- 1.2% in the alpha-stat group). In both groups, the intracranial pressure remained below the critical level of 25 mm Hg, even at a retrograde perfusion pressure of 70 mm Hg. Cerebral lactate production was higher in the alpha-stat group than the pH-stat group during retrograde cerebral perfusion at pressures of 10-30 mm Hg. Compared to the alpha-stat strategy, the pH-stat strategy significantly improved brain tissue perfusion. With an open inferior vena cava, the optimal perfusion pressure seems to be 40-50 mm Hg.

Animals↗

PubMed2005

alpha-Glucosidase from a strain of deep-sea Geobacillus: a potential enzyme for the biosynthesis of complex carbohydrates.

An alpha-glucosidase from Geobacillus sp. strain HTA-462, one of the deepest sea bacteria isolated from the sediment of the Mariana Trench, was purified to homogeneity and estimated to be a 65-kDa protein by SDS-PAGE. At low ion strength, the enzyme exists in the homodimeric form (130 kDa). It is a thermo- and alkaline-stable enzyme with a half-life of 13.4 h and a maximum hydrolytic activity at 60 degrees C and pH 9.0 in 15 mM glycine-NaOH buffer. The enzyme exclusively hydrolyzed alpha-1,4-glycosidic linkages of oligosaccharides in an exo-type manner. The enzyme had an overwhelming transglycosylation activity and glycosylated various non-sugar molecules when maltose was used as a sugar donor. It converted maltose to isomaltose. The gene encoding the enzyme was cloned and sequenced. The recombinant enzyme could be extracellularly overproduced by Bacillus subtilis harboring its gene and preserved the primary properties of the native enzyme. Site-directed mutagenesis experiments showed that Asp98 is essential for the enzyme activity in addition to Asp199, Asp326, and Glu256.

Amino Acid Sequence↗

PubMed2005

Intravenous delivery of liposome-mediated nonviral DNA is less toxic than intraperitoneal delivery in mice.

Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells. Liposomes have been described as having better efficacy in gene delivery, and an advantage of using liposomes as gene carriers is that they can be used repeatedly in vivo. The objective of this study is to compare the effect of gene delivery routes and to determine whether systemic delivery of the rat insulin promoter (RIP)-directed suicide gene construct would permit cell-specific gene delivery in vivo. Severe combined immunodeficient (SCID) mice were injected with liposome-RIP-TK (thymidine kinase) complex by either the intraperitoneal or the intravenous route. Twenty-four hours post gene delivery, mice received ganciclovir (GCV) treatment twice daily for 14 days. Mice were sacrificed at various time points. Complete necropsy and serum chemistry analysis were performed. Islet morphology was determined using hematoxylin and eosin (H&E) staining. Serum glucose and insulin levels were also determined. To determine the toxic effect on pancreatic islet cells, immunostaining of insulin-producing and glucagon-producing cells was carried out at each time point. H&E staining indicated that both intravenous and intraperitoneal liposome-RIP-TK gene expression had no effect in normal endocrine islet cells. Both gene-delivery routes in mice resulted in normal glycemia and serum insulin levels. The endocrine islets were intact, with a normal distribution pattern of insulin-producing beta cells and glucagon-secreting alpha cells. However, serum chemistry analysis revealed significantly elevated levels of liver enzymes; suggesting that possible liver damage had occurred with the intraperitoneal gene delivery of liposome-pRIP-TK. Intravenous liposome-mediated gene delivery had no effect on liver enzyme levels. Liposome-mediated gene delivery via intravenous injection was less toxic than intraperitoneal delivery. This gene-delivery route requires fewer liposome-DNA complexes and maintains normal liver function. Thus, intravenous delivery of gene therapy would be superior to intraperitoneal administration of gene therapy in mice.

Animals↗

PubMed2005

An alkali-inducible flotillin-like protein from Bacillus halodurans C-125.

Flotillins are markers of lipid microdomains, and have emerged as a key concept in cellular biology. However, it remains unclear whether flotillin proteins exist in prokaryotic cells. The amino acid sequence of the BH3500 protein from Bacillus halodurans was 30% identical to that of flotillin-1. Motif analysis revealed that several specific residues (SPFH and flotillin domains, an AEA-repeat structure) and five potential phosphorylation sites are conserved in the BH3500 protein. In addition, the BH3500 protein was found to possess two transmembrane-spanning domains at the N-terminus, which is consistent with the common properties of flotillin-1. The BH3500 protein was detected in the Triton-insoluble, buoyant membrane fraction of B. halodurans by mass spectrometry and Western blotting. Interestingly, BH3500 was expressed strongly in alkaline conditions at both transcriptional and translational levels, which implies that it is one of the alkali-inducible proteins.

Alkalies↗

PubMed2005

[Maize growth and phosphorous- and zinc uptake under different phosphorous supply levels].

By the method of solution culture, this paper studied the P and Zn uptake of maize at its growth stages under different P supply levels. The results showed that maize growth reached its maximum at the supply level of 100 micromol P.L(-1), while the root/shoot ratio was the highest at 0.1 micromol.L(-1) P supply. Increase of P concentration in culture solution promoted the uptake of P, but decreased its utilization rate. The Zn content in root increased with increasing P supply, while that in shoot varied little, which meant that the Zn transportation rate from root to shoot had little change with increasing P concentration. There was a positive correlation between the concentrations of Zn and P in maize seedling roots.

Phosphorus↗

PubMed2004

Cloning, expression, and characterization of a glycoside hydrolase family 86 beta-agarase from a deep-sea Microbulbifer-like isolate.

The gene for a novel beta-agarase from a deep-sea Microbulbifer-like isolate was cloned and sequenced. It encoded a mature protein of 126,921 Da (1146 amino acids), which was a modular protein including two tandem carbohydrate-binding module (CBM)-like sequences and a catalytic module. The catalytic module resembled a glycoside hydrolase family 86 beta-agarase, AgrA, from Pseudoalteromonas atlantica T6c with 31% amino acid identity. Its recombinant agarase was hyper-produced extracellularly using Bacillus subtilis as the host and purified to homogeneity. The activity and stability were strongly enhanced by CaCl2. The maximal enzyme activity was observed at 45 degrees C and pH 7.5 in the presence of 10 mM CaCl2. The enzyme was an endo-type beta-agarase and degraded agarose and agarose oligosaccharides more polymerized than hexamers to yield neoagarohexaose as the main product. This is the first glycoside hydrolase family 86 enzyme to be homogeneously purified and characterized.

Alteromonadaceae↗

PubMed2004

Mass-spectrometric identification of binding proteins of Mr 25,000 protein, a part of vitellogenin B1, detected in particulate fraction of Xenopus laevis oocytes.

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.

Animals↗

PubMed2004

Use of a pH-stat strategy during retrograde cerebral perfusion improves cerebral perfusion and tissue oxygenation.

BACKGROUND: Although it is well documented that the use of a pH-stat strategy during hypothermic cardiopulmonary bypass improves cerebral blood flow, an alpha-stat strategy has been almost exclusively used during retrograde cerebral perfusion. We investigated the effects of pH-stat and alpha-stat management on brain tissue blood flow and oxygenation during retrograde cerebral perfusion in a porcine model to determine if the use of a pH-stat strategy during retrograde cerebral perfusion improves brain tissue perfusion. METHODS: Fourteen pigs were managed by an alpha-stat strategy (alpha-stat group, n = 7) or by a pH-stat strategy (pH-stat group, n = 7) during 120 minutes of hypothermic retrograde cerebral perfusion. Retrograde cerebral perfusion was established through the superior vena cava. Brain tissue blood flow and oxygenation were measured continuously with a laser flowmeter and near infrared spectroscopy, respectively. Brain tissue water content was determined at the end of the experiments. RESULTS: During cooling, brain tissue blood flow was significantly higher with use of the pH-stat strategy than with the alpha-stat strategy (86% +/- 10% versus 40% +/- 3% of baseline). During retrograde cerebral perfusion, brain tissue blood flow was also significantly higher (about three times higher) in the pH-stat group than in the alpha-stat group (15% +/- 4% versus 5% +/- 1% of baseline at 60 minutes of retrograde cerebral perfusion). Tissue oxygen saturation appeared to be higher during retrograde cerebral perfusion in the pH-stat group than in the alpha-stat group. Brain tissue blood flow during rewarming remained significantly higher with the use of pH-stat than with the use of alpha-stat. Brain tissue water contents were similar in both groups. CONCLUSIONS: In our pig model, the use of a pH-stat strategy during retrograde cerebral perfusion significantly improves brain tissue perfusion. Therefore, to improve retrograde cerebral blood flow during retrograde cerebral perfusion, it may be preferable to use a pH-stat strategy, rather than an alpha-stat strategy.

Acid-Base Equilibrium↗

PubMed2004

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94.

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).

Alteromonadaceae↗

PubMed2003

Spatial approximation between the amino terminus of a peptide agonist and the top of the sixth transmembrane segment of the secretin receptor.

Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-l-phenylalanine into the position of His(1) of rat secretin ([Bpa(1),Tyr(10)]secretin-27). Because His(1) is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa(-1),Tyr(10)]secretin-27) and -2 (rat [Bpa(-2),Gly(-1),Tyr(10)]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation.

Amino Acid Sequence↗

PubMed2003

Spatial approximation between two residues in the mid-region of secretin and the amino terminus of its receptor. Incorporation of seven sets of such constraints into a three-dimensional model of the agonist-bound secretin receptor.

Photoaffinity labeling of receptors by bound agonists can provide important spatial constraints for molecular modeling of activated receptor complexes. Secretin is a 27-residue peptide hormone with a diffuse pharmacophoric domain that binds to the secretin receptor, a prototypic member of the Class B family of G protein-coupled receptors. In this work, we have developed, characterized, and applied two new photolabile probes for this receptor, with sites for covalent attachment in peptide positions 12 and 14, surrounding the previously most informative site of affinity labeling of this receptor. The [Tyr10,(BzBz)Lys12]rat secretin-27 probe covalently labeled receptor residue Val6, whereas the [Tyr10,(BzBz)Lys14]rat secretin-27 probe labeled receptor residue Pro38. When combined with previous photoaffinity labeling data, there are now seven independent sets of constraints distributed throughout the peptide and receptor amino-terminal domain that can be used together to generate a new molecular model of the ligand-occupied secretin receptor. The amino-terminal domain of this receptor presented a stable platform for peptide ligand interaction, with the amino terminus of the peptide hormone extended toward the transmembrane helix domain of the receptor. This provides clear insights into the molecular basis of natural ligand binding and supplies testable hypotheses regarding the molecular basis of activation of this receptor.

Amino Acid Sequence↗