Transcriptome survey of phototransduction and clock genes in marine bivalves.

Marine animals exhibit a variety of biological rhythms, such as solar and lunar-related cycles; however, our current molecular understanding of biological rhythms in marine animals is quite limited. Identifying and understanding the expression patterns of clock genes from available transcriptomes will help elucidate biological rhythms in marine species. Here, we perform a comprehensive survey of phototransduction and circadian genes using the mantle transcriptome of the scallop Patinopecten yessoensis and compare the results with those from three other bivalves. The comparison reveals the presence of transcripts for most of the core members of the phototransduction and circadian networks seen in terrestrial model species in the four marine bivalves. Matches were found for all 37 queried genes, and the expressed transcripts from the deep sequencing data matched 8 key insect and mammalian circadian genes. This demonstrates the high level of conservation of the timekeeping mechanism from terrestrial species to marine bivalves. The results provide a valuable gene resource for studies of "marine rhythms" and also further our understanding of the diversification and evolution of rhythms in marine species.

Novel polymorphic microsatellite markers isolated from the pen shell Atrina pectinata (Mollusca: Bivalvia: Pinnidae).

In this study, we isolated 21 novel polymorphic microsatellite DNA loci from the pen shell Atrina pectinata using magnetic-bead hybridization enrichment. The characteristics of these loci were tested using a population of 30 individuals collected from the Penglai coast, Shandong Province. The number of alleles ranged from 2 to 13, and polymorphism information content (PIC) varied from 0.1730 to 0.8954. Values for observed heterozygosity (HO) and expected heterozygosity (HE) ranged from 0.0714 to 0.9231 and from 0.1948 to 0.9237, respectively. Four loci deviated significantly from Hardy-Weinberg equilibrium. The newly developed microsatellite markers will be beneficial in assessing the genetic diversity, population structure and genetic conservation of A. pectinata, and in other relevant research.

Isolation and characterization of 48 polymorphic microsatellite markers for the blood clam Scapharca broughtonii (Arcidae).

Blood clams (Scapharca broughtonii) are widely cultivated and consumed in noutheast Asia. Forty-eight polymorphic microsatellite loci were developed for this clam using magnetic-bead hybridization enrichment. The number of alleles per locus ranged from 2 to 14. Polymorphism of these loci was assessed in 30 individuals from a population collected from coastal areas of Qingdao, China. The values of observed heterozygosity, expected heterozygosity and polymorphism information content per locus ranged from 0.1034 to 0.9655, from 0.1831 to 0.9208, and from 0.1638 to 0.8964, respectively. Forty-three of 48 loci conformed to Hardy-Weinberg equilibrium. These microsatellite loci would be useful for molecular genetic breeding, population genetics, genome mapping, and other relevant research on S. broughtonii.

Genetic susceptibility to haemorrhagic fever with renal syndrome caused by Hantaan virus in Chinese Han population.

We report a significantly higher occurrence of HLA-DRB1*09 (51% vs. 23%, P = 0.002, OR = 3.57) and HLA-B*46-DRB1*09 (26% vs. 8%, P = 0.018, OR = 3.76) in patients with haemorrhagic fever with renal syndrome (HFRS) compared to the control group, suggesting Hantaan virus-induced HFRS is associated with a genetic predisposition in the Chinese Han population.

Localization of CD226 in the mouse hippocampus and cerebellum during adulthood and postnatal development.

CD226, a member of cell adhesion molecules, has been widely studied in the immune system; however, its expression in the CNS remains unknown. In our present study, we detected CD226 mRNA and protein in the mouse hippocampus and cerebellum by RT-PCR and Western blotting, respectively. Immunohistochemical studies found that CD226 is primarily located in the hilus of the dentate gyrus and stratum lucidum aligned along the pyramidal cells in the hippocampal CA3 area, the interspaces of granular cells and the somata of the Purkinje cells in the cerebellar cortex during adulthood. Double-staining results revealed that CD226 co-localized well with synaptic marker proteins including synaptophysin, syntaxin and PSD-95. During postnatal development, CD226 could not be detected at its adult locations until postnatal day 12; however, it was temporally expressed in the somata of neighboring or distant nuclei associated with its adult location. These results showed the diverse localization of CD226 in the mouse hippocampus and cerebellum for the first time and suggested its potential role in the CNS.

An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells.

Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells.

Selective proapoptotic activity of a secreted recombinant antibody/AIF fusion protein in carcinomas overexpressing HER2.

Apoptosis-inducing factor (AIF) represents a caspase-independent apoptotic pathway in the cell, and a mitochondrial localization sequence-truncated AIF (AIFDelta1-120) can be relocated from the cytoplasm to the nucleus and exhibit a constitutive proapoptotic activity. Here, we generated a chimeric immuno-AIF protein, which comprised an HER2 antibody, a Pseudomonas exotoxin translocation domain and AIFDelta1-120. Human Jurkat cells transfected with the immuno-AIF gene could express and secrete the chimeric protein, which selectively recognized HER2-overexpressing tumor cells and was endocytosed. Subsequent cleavage of truncated AIF from immuno-AIF and its release from the internalized vesicles resulted in apoptosis of tumor cells. Intramuscular injection of the immuno-AIF gene caused significant suppression of tumors and substantially prolonged mice survival in an HER2-overexpressing xenograft tumor model. Our study demonstrates the feasibility of the immuno-AIF gene as a novel approach to treating cancers that overexpress HER2.

Improved PCR-based subtractive hybridization strategy for cloning differentially expressed genes.

An improved PCR-based subtractive hybridization strategy was used to clone apoptosis-related genes induced by all-trans retinoic acid (ATRA) from human promyelocytic leukemia cell line HL-60 cells. The protocol used the cap-finder method, long-distance PCR, streptavidin magnetic bead-mediated subtraction and spin column chromatography. Twenty-seven clones related to apoptosis were identified by reverse dot blot assay. Seventeen were known genes, of which seven have been reported to be apoptosis related. The remaining 10 were unknown genes, five of which were sequenced and named apr-1 to apr-5. apr-1, apr-2, apr-3 and TNF were reidentified by reverse dot blot, and it is suggested that they might be related to apoptosis. The results suggest that this strategy might be efficient for large-scale cloning of differentially expressed genes in target cells.

Genetic co-inactivation of macrophage- and T-tropic HIV-1 chemokine coreceptors CCR-5 and CXCR-4 by intrakines.

CC-chemokine receptor (CCR)-5 is the principal coreceptor for the entry of macrophage (M)-tropic HIV-1 viruses into a cell, while CXC-chemokine receptor (CXCR)-4 is the principal coreceptor for T cell line (T)-tropic HIV-1. In this study, we utilized a novel intracellular chemokine (intrakine) strategy to co-inactivate genetically both CCR-5 and CXCR-4 in human lymphocytes. The principle of co-inactivation of CCR-5 and CXCR-4 was illustrated by targeting the CC-intrakine and CXC-intrakine to the lumen of the endoplasmic reticulum (ER) for intracellular blockade of the transport of newly synthesized chemokine coreceptors to the cell surface. The lymphocytes with the phenotypic knock-out of CCR-5 and CXCR-4 were found broadly to resist the infection of M-tropic, T-tropic and dual-tropic HIV-1 viruses. Moreover, the transduced lymphocytes retained normal cell features, including the responsiveness to mitogen and recall antigen stimulation. Thus, this study to our knowledge, is the first to demonstrate that genetic co-inactivation of the M- and T-tropic HIV-1 principal coreceptors in lymphocytes or other cells could be a viable strategy for the long-term control of HIV-1 infection.

Anti-HIV type 1 activity of wild-type and functional defective RANTES intrakine in primary human lymphocytes.

We have developed a genetic "intrakine" strategy to inactivate the CC-chemokine receptor 5 (CCR-5), the principal coreceptor for macrophage (M)-tropic HIV-1 viruses (Yang et al, 1997). The inactivation of CCR5 was achieved by targeting a modified CC-chemokine (RANTES) to the lumen of the endoplasmic reticulum (ER) to block the transport of the newly synthesized CCR-5. The transduced lymphocytes with the phenotypic CCR5 knockout were shown to be resistant to M-tropic HIV-1 infection. This study illustrated the feasibility of the intrakine strategy to block HIV-1 infection. In our current study, the potential clinical application of the intrakine approach was further evaluated in human peripheral blood lymphocytes (PBLs). PBLs were transduced with the RANTES intrakine gene by using retroviral vectors with the truncated low-affinity human nerve growth factor receptor (deltaNGFR) marker, and then isolated by an anti-NGFR antibody/magnetic bead method. The surface expression of CCR-5 in the transduced lymphocytes was dramatically inhibited, as demonstrated by flow cytometric assays. The transduced PBLs were shown to resist various types of M-tropic HIV-1 virus infection. The cell viability, cell proliferation rates, and cell surface markers of the intrakine-transduced PBLs were shown to be comparable to those of control PBLs. The transduced PBLs were also found to respond to the stimulation of various CXC- and CC-chemokines, other than RANTES. The transduced PBLs responded to tetanus antigen stimulation by increasing IL-2 production and cell proliferation. In addition, a functionally defective mutant of RANTES that retains its binding activity to CCR-5, but loses its signaling ability, was used to generate a mutant RANTES intrakine. The primary lymphocytes transduced with the mutant RANTES intrakine were found to be resistant to M-tropic HIV-1 infection. From these results, we conclude that the primary human lymphocytes transduced with either the wild-type or functionally defective RANTES intrakine are resistant to M-tropic HIV-1 infection, and maintain their basic biological functions. This study, therefore, indicates the potential clinical application of the intrakine approach for HIV-1 gene therapy.

Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection.

A genetic defect in a CC-chemokine receptor (CCR)-5, the principal coreceptor for the macrophage-tropic HIV type 1 (HIV-1), recently was found to naturally protect CCR-5-defective, but healthy, individuals from HIV-1 infection. In this study, we mimic the natural resistance of the CCR-5-defective individuals by designing a strategy to phenotypically knock out CCR-5. The inactivation of the CCR-5 coreceptor is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5. The lymphocytes transduced to express the intracellular chemokine, termed "intrakine," were found to be viable and resistant to macrophage-tropic HIV-1 infection. Thus, this gene-based intrakine strategy targeted at the conserved cellular receptor for the prevention of HIV-1 entry should have significant advantages over currently described approaches for HIV-1 therapy.

Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy.

CXC-chemokine receptor (CXCR)-4/fusin, a newly discovered co-receptor for T-cell line (T)-tropic HIV-1 virus, plays a critical role in T-tropic virus fusion and entry into permissive cells. The occurrence of T-tropic HIV viruses is associated with CD4-positive cell decline and progression to AIDS, suggesting that the T-tropic HIV-1 contributes to AIDS pathogenesis. In this study, we used a novel strategy to inactivate CXCR-4 by targeting a modified CXC-chemokine to the endoplasmic reticulum (ER) to block the surface expression of newly synthesized CXCR-4. The genetically modified lymphocytes expressing this intracellular chemokine, termed "intrakine", are immune to T-tropic virus infection and appear to retain normal biological features. Thus, this genetic intrakine strategy is uniquely targeted at the conserved cellular receptor for the prevention of HIV-1 entry and may be developed into an effective treatment for HIV-1 infection and AIDS.

A new class of antigen-specific killer cells.

A new class of antigen-specific killer cells that combines the features of antibody-mediated and cell-mediated immunity was designed. The principle and feasibility of this strategy are illustrated by creating an antigen-specific killer cell to produce and secrete targeted antibody-toxin molecules that recognize and kill human immunodeficiency virus (HIV)-1-infected cells. The transduced lymphocytes, which were able to produce and secrete the targeted toxin proteins, remained viable due to the lack of the target antigen on the cell surface. These transduced cells were found to have selective and potent cytotoxicity to the HIV-infected cells. This approach combines the specificity of antibodies, extreme potency of toxins, and effector-cell properties of T-cells to generate a new class of potent antigen-specific killer cells, which may have broad applications for the treatment of viral infection and other diseases. This study demonstrates the principle that mammalian cells can be genetically modified to produce targeted toxins, indicating that in vivo production of targeted toxins can be achieved to locally or systematically destroy targeted cells.

Potent antitumour activity of a new class of tumour-specific killer cells.

Two approaches to the antibody-directed targeting of toxic or cytolytic activity and augmentation of cellular immune responses have been explored for tumour immunotherapy, but so far success has been limited. Obstacles facing immunotherapy are the limited accessibility of antibodies or antibody conjugates to solid tumours and the difficulty in obtaining tumour-specific cytotoxic lymphocytes. Here we generate a new class of tumour-specific killer cells by genetically modifying lymphocytes to produce and secrete a targeted toxin against an oncoprotein overexpressed on breast and other tumour cells. The transduced lymphocytes were shown to have potent and selective cytotoxicity to tumours in culture and nude mouse models. The potent in vivo antitumour activity is probably a result of the migration of the lymphocytes to tumours as a targeted toxin carrier, and production and accumulation of the targeted toxins inside tumours as a producer. Our approach, which has features of both antibody-directed and cell-mediated immunotherapy, may have application in a gene therapy context.

Intra- and extracellular immunization against HIV-1 infection with lymphocytes transduced with an AAV vector expressing a human anti-gp120 antibody.

Recently, we developed a novel anti-HIV-1 approach by transducing an anti-gp120 antibody gene into lymphocytes, resulting in the resistance to HIV-1 infection by the combined intra- and extracellular binding activities of the neutralizing antibody. To extend this study, we improved the co-expression of the heavy and light chains of the Fab105 fragment of the anti-gp120 antibody F105 by using an internal ribosome entry site (IRES) sequence. The Fab105 expression cassette was then cloned into an adeno-associated virus (AAV) shuttle vector, and encapsidated recombinant AAV-Fab105 vectors were produced. The Fab105 antibody gene was shown to be transduced into human lymphocytes by using the recombinant AAV viruses. The transduced lymphocytes were able to produce and secrete the Fab105 fragments, while maintaining their normal morphology, growth rates, and responsiveness to mitogen stimulation. The infection of several primary HIV-1 patient isolates was effectively blocked in the transduced lymphocytes. This study indicates that the combined intra- and extracellular immunization approach may be useful for the treatment of HIV-1-infected patients.